The cosmid library of rat genomic DNA was screened with the coding region of microsomal stearoyl-CoA desaturase (SCD) cDNA as the probe. Two independent clones having 36 kb (SCDI) and 31 kb (SCDII) of inserts were isolated. Southern mapping analyses revealed that SCDI codes for SCD and SCDII for an SCD-homologue, and that the genes differ markedly in the upstream regulatory regions. Nucleotide sequence analysis of SCDI revealed that the SCD gene is organized with 6 exons and 5 introns spanning about 15 kb, thus coding an mRNA with an open reading frame of 1,074 bp of the desaturase. Transcription starts at 114 by upstream of the translation initiation codon, and 32 bp upstream of the transcription initiation site there is a TATA box. A sequence similar to Fat Specific Element 2 (FSE2), the negative regulatory element of adipocyte differentiation, was present at 45 bp upstream of the TATA box. The tissue distribution and induction due to dietary manipulation of SCDI and SCDII mRNAs as revealed by Northern hybridization were markedly different from each other.