Raising uncastrated male pigs could have significant economic benefits for pig production. Uncastrated male pigs can accumulate high levels of 16-androstene steroids, however, resulting in boar taint, which is highly objectionable to consumers. Cytochrome P450-c17 (CYP17) interacts with cytochrome b5 in the biosynthesis of the 16-androstene steroids and the sex steroids from pregnenolone. Amino acid substitutions in CYP17 could therefore affect the ability of this enzyme to catalyze the reactions leading to the production of androstenone and the sex steroids. In this study, we established a sensitive and flexible single-stranded conformational polymorphism technique capable of detecting a single nucleotide polymorphism. We then used this method to identify a substitution from T to A at nucleotide 1317 of CYP17, which caused a change in the amino acid sequence from Leu(439) to His(439). This mutation, however, did not alter the enzyme activity of CYP17 in the biosynthesis of androstenone or sex steroids. Other polymorphisms previously suggested for CYP17, which are vital for the functional interaction of CYP17 with CYB5 in human, were not observed. This study suggests that the synthesis of androstenone in pig testis is not directly affected by any polymorphisms in the coding region of the porcine CYP17 gene.