The onset and localization of P450(17-alpha)-hydroxylase (P450(17-alpha)) and P450 aromatase (P450arom) mRNA expression were studied in the gonads of chicken embryos at Days 4-9 of incubation by in situ hybridization analysis, and expression of both mRNAs was measured in the ovary and testis of the chicken after sexual differentiation by Northern and slot-blot analysis and was related to changes in concentration of 17-beta-estradiol (E2) in the gonad. In situ hybridization analysis showed the first detection of P450(17-alpha) mRNA at Days 5-6 of incubation in the genetic male and female gonads and the first detection of P450arom at Day 6.5 of incubation in the female gonad but none was detected in the male gonad. Both mRNAs were observed in the medullary cords of the gonad. Slot-blot analysis demonstrated that levels of P450(17-alpha) mRNA in the ovary remained high during the period between Day 10 of incubation and 7 days of posthatching, while those in the testis remained low at Days 10-14 of incubation but increased at Day 16 of incubation up to posthatching days. Levels of P450arom mRNA in the left ovary were invariably higher than those of the right ovary and testis throughout the period between Day 10 of incubation and 7 days of posthatching. These results suggest that expression of P450arom mRNA occurring in the genetic female gonad around Day 6 is associated, at least in a part, with sexual differentiation toward femaleness by increasing E2 production which may promote cellular proliferation of the left gonad. In contrast, in the genetic male gonad marked expression of P450(17-alpha) mRNA with little expression of P450arom mRNA may lead to production of androgen which promotes the gonadal development to the testis.