The aim of the present study is the isolation of subpopulations of steroid-producing cells in the ovary of the newly hatched chicken. Cells were obtained by fractional trypsin dissociation of the ovary and isopycnic separation in a continuous metrizamide gradient (0–30%). Testosterone and 17β-estradiol secretion was measured by radioimmunoassay in the incubation medium of the isolated cells. Six fractions of ovarian cells were studied. Fraction II (density 1.080) contained typical steroidogenic cells with a positive reaction for 3β-hydroxysteroid dehydrogenase, Δ 5-isomerase. This fraction secreted testosterone (3.7 ng/10 6 cells/2 hr) but no 17β-estradiol secretion was detectable. The majority (85%) of cells obtained in fraction VI (density 1.150) were relatively undifferentiated and contained polyribosomes, mitochondria with lamellar cristae, and few rough endoplasmic reticulum; only 3–5% of the cells of this fraction were similar to those of fraction II. In fraction VI the highest level of 17β-estradiol secretion was found (2.9 ng/10 6 cells/2 hr) whereas testosterone was at a minimum level (0.06 ng/10 6 cells/2 hr). Results strongly suggest the existence of two cell subpopulations in the inmature chicken ovary: typical steroidogenic and poorly differentiated cells which secrete testosterone and 17β-estradiol, respectively.