Specific receptors and metabolic responses to luteinizing hormone (LH) were analyzed in testicular Leydig cells purified by centrifugation of collagenase-dispersed rat interstitial cells on density gradients of 14-32% Metrizamide. This procedure separated the interstitial cells into an upper, poorly responsive fraction and a lower, more dense population with high LH receptor content and prominent cyclic AMP and testosterone responses of gonadotropic stimulation. The upper layer consisted of morphologically heterogeneous and extensively vacuolated cells, of which relatively few were structurally identifiable as Leydig cells. The lower layer was comprised of almost homogenous Leydig cells when analyzed by electron microscopy and autoradiography, and sedimented as two adjacent bands with similar hormonal responses and densities of 1.085 and 1.105 g/cm3. The lighter and less responsive cell population appeared to result from the presence of damaged cells in the interstitial cell preparation and could be removed during density gradient isolation of the homogeneous and biologically active Leydig cell fraction. The more dense and active Leydig cell population retained hormonal responsiveness during culture for 24 h and showed loss of LH receptors and low concentrations of gonadotropin in vitro. These findings emphasize the importance of appropriate fractionation of rat interstitial cells to isolate the structurally intact and functionally active population of Leydig cells during studies on LH receptors and section in the testis.