A complete amino acid sequence for rat testis P-450(17)alpha was deduced from nucleotide analysis of a cDNA clone isolated from a rat Leydig cell cDNA library. This DNA clone, containing initiation and termination codons and a polyA tail, translated a polypeptide in COS-1 cells that expressed both 17 alpha-hydroxylase and 17,20 lyase activities. It exhibited significant similarity to the nucleotide and deduced amino acid sequences of the bovine and human cytochrome P-450(17)alpha, particularly with respect to the highly conserved regions and secondary structure. The P-450(17)alpha appears to be anchored to the membrane of the endoplasmic reticulum through two transmembrane regions, specifically the N terminal insertion peptide and the stop-transfer sequence. Hydropathic analysis indicates that the remainder of the C terminus is associated with the membrane through four hydrophobic clefts, including the putative steroid binding site.