Whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor levels correlate with the rapid in vivo growth rate of the testes in the prepubertal rat was examined. Low salt chromatin-localized 1,25-(OH)2D3 receptors were compared in the testes and intestinal mucosa (control) of prepubertal, peripubertal, and mature rats (37, 49, and 90 days old, respectively). The number of 1,25-(OH)2D3 receptors per g wet wt was significantly (P less than 0.02) reduced in the testes of the prepubertal rats compared to those in the peripubertal and mature groups. Conversely, no changes were observed in the 1,25-(OH)2D3 receptor levels in the control tissue intestinal mucosa among these age groups. Further experiments confirmed the identity of the testicular 1,25-(OH)2D3 receptors. The specific [3H]1,25-(OH)2D3-binding component was predominantly localized in the nuclei/chromatin fraction in hypotonic buffers. Scatchard analysis of [3H]1,25-(OH)2D3 binding to the testicular chromatin of adult rats yielded a single specific binding component with a Kd of 0.33 +/- 0.06 nM and a Nmax of 102.3 +/- 6.4 fmol/g tissue (n = 6), which was inhibited by excess 1,25-(OH)2D3, but only minimally by 50 nM 25-hydroxyvitamin D3. Sucrose gradient analysis required hydroxylapatite treatment of fractions after centrifugation to remove free 3H-labeled steroid. With this modification, a discrete 3.6S peak of [3H]1,25-(OH)2D3 was unmasked, which was eliminated by excess 1,25-(OH)2D3, but not by 50 nM 25-hydroxyvitamin D3, or 1 microM cortisol, or the progesterone analog promegestone. In spite of its seemingly ubiquitous distribution, the 1,25-(OH)2D3 receptor does exhibit tissue specificity, since it appears to be absent in the prostate and, at best, greatly reduced in the epididymis. The cellular localization of the testicular 1,25-(OH)2D3 receptors was examined by mechanically separating interstitial cells (93.7% of the total [125I]hCG binding) from the tubules. Under these conditions, 91.3% of the specific [3H]1,25-(OH)2D3 binding occurred in the tubular chromatin preparation. Thus, these data provide evidence for the presence of a specific 1,25-(OH)2D3 receptor in the seminiferous tubules of the rat testis. Moreover, the temporal correlation of increased 1,25-(OH)2D3 receptor levels with testicular maturation suggests a better correlation to testicular function and spermatogenesis than to growth of the organ in vivo.