The purpose of the present study is to provide further evidence in support of the theory which suggests that two cells are involved in the secretion of estrogens and androgens in the theca of the preovulatory ovarian follicles in the domestic fowl. The third (F3), the fourth (F4), and the fifth (F5), in order of decreasing size follicles, were obtained from white Leghorn hens. Theca cells were dispersed by trypsin treatment, and isolated cells were centrifuged in a continuous Percoll gradient (0–100%). Testosterone and 17β-estradiol secretion from cell samples in each density gradient fraction were radioimmunologically assayed. The highest secretion of 17β-estradiol was registered in cells isolated in density fractions ranging from 1.059 to 1.070 g/ml, while the maximal testosterone production was found in fractions from 1.037 to 1.048 g/ml. The different results were obtained from the three types of follicles studied (F3–F5). Morphological study of the F4 follicle showed typical steroidogenic cells with abundant lipid droplets in the cytoplasm in the theca interna, whereas clusters of poorly differentiated epithelial cells were visualized between fibroblast layers of the theca externa. The secretion of aromatizable androgens by the typical steroidogenic cells of the theca interna and estrogen production in the epithelial cells of the theca externa are postulated.