To determine the kinetic characteristics of gonadotropin-induced steroidogenesis and secretion, acute preparations of collagenase-dispersed 55- to 60-day-old rat Leydig cells were suspended in a 2-ml column of Bio-Gel P2 and perifused at a constant rate of 0.5 ml/min with medium 199-0.1% bovine serum albumin at 35 C. Suspended Leydig cells (3-10 × 106) had a limited functional timespan when assessed on the basis of gonadotropin-stimulated testosterone (T) output. Cells were resistant to gonadotropin during both the initial 40 min and after 240 min of perifusion. The basal T output started high, became steady between 40–180 min, and thereafter declined to undetectable levels. Ninety percent of the cells remained trypan blue negative after 240 min of column suspension. During the functional period, rapid and large increases in T release could be induced by a variety of gonadotropins with interstitial cell-stimulating activity in addition to 8-bromo-cAMP. The time delay between gonadotropin contact with the Leydig cell surface and a detectable increase in T output averaged 10 min at infusion concentrations of 1 ng/ml. The decline in the T response after a 16-min exposure to hCG differed considerably from that of LH (ovine or human). A 50% decline in T output occurred 10 min after the peak induced by LH, whereas more than 80 min were required for a similar reduction after hCG. Hence, response curves for LH and cAMP were almost Gaussian in appearance, whereas those for hCG were highly skewed. Repeated exposure to gonadotropin resulted in reduced T responses in all of the systems investigated. At such times of gonadotropin resistance, it was not possible to restore T output by infusing cAMP, indicating postreceptor blockade of T production. The short time period of gonadotropin exposure necessary to induce this gonadotropin resistance or desensitization suggested that receptor down-regulation may not be an important determinant of this postreceptor steroidogenic blockade. These data demonstrate the application of a column perfusion technique to collagenase-dispersed rat Leydig cells, and the marked difference in kinetics of the T responses were in keeping with the known dissociation rates for hCG and LH. In addition, rapid induction of desensitization in vitro occurred in the suspended Leydig cells, indicating that the technique will be valid for the further evaluation of LH receptor mechanisms.