Vacuolar H+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an impor- tant role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To ana- lyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5′-upstream sequence, and the 3′-RACE technique was used to clone the 3′-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5′-UTR, an ORF of 1,134 bp, and a 196 bp 3′-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high ho- mology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expres- sion pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation. Vacuolar H + -ATPase was considered as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in the polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an impor- tant role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. Ana lyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5 ’ The full length of the target clone was 1,340 bp, including a 10 bp 5’-UTR, an The ORF of 1,134 bp, and a 196 bp 3’-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high ho- mology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expres- sion pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with In addition, in vitro ovule culture experiment said that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA expression of GhDET3 increased in transgenic FBP7 :: GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.