The purpose of the present investigation was to measure the concentrations of progesterone (P4), testosterone (T) and estradiol-17 beta (E2) in isolated theca and granulosa layers of the five preovulatory follicles of the domestic hen. The largest follicle (F1), the second largest (F2), third largest (F3), fourth largest (F4) and fifth largest (F5) follicles were removed at 24, 18, 12, 6 and 2 h before the expected ovulation. Theca and granulosa layers were isolated and P4, T, E2 and protein concentrations determined. Protein concentrations of the granulosa and theca layers increased 5- and 15-fold, respectively, during the five ovulatory cycles prior to ovulation. As the follicle approached ovulation, there was a linear decrease in E2 concentration of the theca layer with the most significant decrease (P less than 0.001) occurring between 24 and 18 h of the ovulatory cycle. In the F3 and F4 theca layers, there was a significant increase (P less than 0.005) in E2 at 6 h of the ovulatory cycle. Fluctuations in T concentrations in theca and granulosa layers were similar. There was a significant increase (P less than 0.05) in T in both layers of the F2, F3 and F4 follicles at 6 h followed by a decrease (P less than 0.005) in the theca layers at 2 h of the ovulatory cycle. The P4 concentration of the granulosa layer increased gradually during follicular maturation, with the greatest increase occurring in the F2 and F1 granulosa layers between 18 and 12 h of the ovulatory cycle.(ABSTRACT TRUNCATED AT 250 WORDS)