Over 75% of fatty acids in plants are unsaturated by desaturase enzymes in chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases designated FAD2 and FAD3 primarily desaturate extrachloroplast lipids and occur as integral membrane proteins in the endoplasmic reticulum. The FAD2 gene has been shown by several laboratories to be important in the chilling sensitivity of plants, since polyunsaturated phospholipids are essential for maintaining plant viability at lowered temperatures. Cotton genomic libraries harbored in lambda phage were screened with an Arabidopsis FAD2 hybridization probe to isolate prospective FAD2 genes, in order to study their regulation of gene expression. Two overlapping genomic clones were found to encompass a FAD2 gene by alkaline blot hybridization and DNA sequence analysis. Since restriction fragments from the cloned DNAs correspond to identically-sized cotton genomic DNA fragments, the cloned DNA fragments represent actual genomic fragments encompassing the FAD2 gene. The protein-coding region of this gene is 1,155 bp, and is continuous with no introns. The deduced amino acid sequence of 384 amino acids of the putative cotton FAD2 polypeptide has a high identity (about 75%) with the deduced amino acid sequences of other plant FAD2 enzymes, such as Arabidopsis FAD2 and soybean FAD2. The cotton FAD2 enzyme has histidine-rich motifs that could serve as potential iron-binding domains for electron transport for the desaturation reaction, similar to other plant FAD2 amino acid sequences. Yeast cells transformed with a plasmid construct containing the cotton FAD2 coding region have an appreciable accumulation of linoleic acid (18:2), not normally present in wild-type yeast cells. Thus, this cotton FAD2 gene is truly functional, since it encodes an enzyme that catalyses the desaturation of oleic acid (18:1) into linoleic acid (18:2). The FAD2 gene has one large intron of 2,967 bp in the 5'-flanking region, only 11 bp upstream from the ATG initiation codon. The presence of a large intron in the 5'-flanking region could be important in the transcriptional regulation of this gene. The FAD2 gene promoter/enhancer motif has a potential TATA basal promoter element and two presumptive basic region-helix-loop-helix (bHLH) motifs with the consensus sequence CANNTG. The bHLH or E box motif has been implicated as a seed-specific regulatory element.