We have found high affinity binding of insulin not only in rat liver and kidney, but also in testis and male sex accessory tissues, prostate, seminal vesicle, and epididymis. We have studied particularly the characteristics of insulin binding in the testis. Membranes sedimenting at 100,000 × g showed the highest binding after 6–20 h of incubation at 0 C. Higher temperatures (15 and 25 C) resulted in lower binding. More than 90% of membrane-bound radioactivity after long incubations at 0 C was eluted at the same position as insulin by Sephadex G-50 chromatography. Membranes could be stored at -80 C for several weeks without loss of activity. Studies on binding specificity showed the following order of competition relative to insulin (100): desalanine insulin (84), proinsulin (2), and d6soctapeptide insulin (1). Other peptidic hormones, LH, FSH, PflL, GH, glucagon, and ACTH-(1–24) were totally ineffective. Scatchard representation of the binding data could be resolved into two components with respective affinity constant (Ka)i of 1.6 × 109 M-1 and 3 × 106 M-1. Testicular high affinity binding in adult rats did not vary after 3 days of starvation. However, it increased with age from 1–6 months. By contrast, in rat liver> this type of binding increased after starvation but decreased slightly at 6 months of age. These results show that testicular insulin receptors are similar to those of the liver but may have a different physiological control.