Concentrations of estradiol (E), testosterone (T), and progesterone (P) and levels of the 1.9-kb and 4.0-kb mRNA encoding P450 17 alpha-hydroxylase (P450 17 alpha) and P450aromatase (P450arom), respectively, were measured in the isolated granulosa, theca interna, and theca externa of the largest preovulatory (F1-F4) follicles of the chicken 20 h and 3 h before expected ovulation of F1. In the granulosa layer, E and T concentrations remained low throughout this period of follicular growth, whereas P concentrations were high 3 h before ovulation in F2 and F1 follicles. E levels were highest in the theca interna and externa of the smallest (F4) follicle, decreasing progressively with follicular growth. T content of theca interna and granulosa was greater in less mature F4-F2 follicles than in the most mature (F1) follicle, whereas theca externa contained little T. P was quantifiable in significant amounts only in granulosa tissue, peaking in F2 and F1 follicles at 3 h before ovulation, at which time plasma levels of steroids also rose significantly. P450 17 alpha was expressed mainly in theca interna, whereas the P450arom mRNA levels found in theca externa reflected the pattern of E in this compartment. Expression of these two genes was very low in granulosa tissue, remaining unchanged during follicular growth. These results indicate that E and T synthesis in the chicken ovary is regulated, at least in part, at mRNA levels of P450 17 alpha and P450arom in the theca interna and theca externa layers during follicular growth.