Cytochrome P450c17 is one of the most critical enzymes in steroidogenic pathway, which is essential in the production of sex steroid hormone(androgens and estrogens). Up to date, there are two P450c17 s (P450c17-I and P450c17-Ⅱ) enciodedencoded by two different genes. P450c17-I possesses two enzyme activities, namely 17α-hydroxylase and 17, 20-lyase, while P450c17-Ⅱ only has 17α-hydroxylase. Under the condition of P450c17-I and P450c17-Ⅱcomplete cDNAs, the spatial and temporal expressions of the two genes are studied by the semi-RT-PCR technology, combined with gonad development and serum testosterone(T) level. The results of gene spatial expression analysis showed that tongue sole P450c17-I was distributed widely, which was expressed in testis, stomach, intestine, heart, spleen and brain, while tongue sole P450c17-Ⅱdistribution was tissue-specific, which was only expressed in testis and head kidney. During various developmental stages of testis, P450c17-I expression pattern revealed developmental stage-dependency, while the expression pattern of P450c17-Ⅱ was on the contrary. It is predicted that, during the whole reproductive period, P450c17-I regulates the sex steroid hormone on the transcriptional level or after transcription in male tongue sole, while P450c17-Ⅱmay be related to progestin in inducing spermiation and DNA replication in spermatogonia.