Most of the available information on Leydig cells has been obtained using a rodent model system. With an objective to extend the observations made with rat Leydig cells (RLCs) to primates, a method has been developed to isolate Leydig cells from monkey (Macaca radiata) testis. Enzymatic dissociation of monkey testis followed by Percoll-gradient fractionation of the interstitial cells resulted in the recovery of Leydig cells at densities corresponding to 1.064-1.070 g/ml. Purified (90-94%) monkey Leydig cells (MLCs) stained positive for the Leydig cell marker 3β-hydroxysteroid dehydrogenase. The cells responded to in vitro addition of human chorionic gonadotropin (hCG) and produced testosterone. Comparison of the in vitro testosterone-producing ability of MLCs with RLCs revealed that MLCs have much less steroidogenic capacity compared with the RLCs. Analysis revealed that limitation in substrate availability to mitochondrial P450 side chain cleavage enzyme and low mitochondrial and smooth endoplasmic reticulum content in MLCs could be the possible reasons for the low steroidogenic capacity of the MLCs.