Binding of insulin to purified intact Leydig cells (LC) and LC membranes, and levels of insulin in plasma and testicular interstitial fluid (IF) were quantitatively evaluated in rats at three stages of development. Specific insulin binding to intact LC increased significantly with age. Scatchard analysis of the binding data always gave curvilinear plots; the number of high affinity binding sites/LC were 2590±514, 3977±701 and 8342±2039 at 21, 40 and 70 days respectively. When the results were expressed per μg membrane protein, the maximal specific insulin binding also increased between 21 and 40 days but did not significantly change thereafter. With ageing, insulin levels in testicular IF decreased (3·05±0·30, 2·48±0·22 and 1·66±0·13 μg/l in 21-, 40- and 70-day-old rats) whereas plasma insulin increased. Taken together, these results suggest (1) that the intratesticular environment in this hormone cannot be evaluated by plasma insulin levels—testicular IF insulin concentration is probably a better index and (2) that insulin may play a role in the development of LC function during sexual maturation.