Collagenase dispersed cells from immature or mature rat testis were separated into 11 bands on a discontinuous Percoll gradient. Erythrocytes sedimented at the bottom. Only three bands (8, 9 and 10) bind efficiently [<sup>125</sup>I]hCG. In immature rats, cells in bands 8, 9 and 10 bind hCG with the same affinity, but differ slightly in their total number of sites (average 65,000 ± 4,000 sites per Leydig cell) and in their cAMP and testosterone production in response to hCG. They present 8 % of the total number of dispersed cells. In mature rat testis, populations 8, 9 and 10 differed markedly in their capacity to bind [<sup>125</sup>I]hCG and in their response to hCG as regards an increase in cAMP and testosterone production. Testosterone production was increased 2- and 17-fold in bands 8 and 10, respectively. In mature rats only 1.2% of the total cells are Leydig cells. In both mature and immature rats, band 10 is made of 100% Leydig cells, as all cells stained positively for 3β-hydroxysteroid dehydrogenase.