The testis is rich in central nervous system-type neuropeptides, including a GH-releasing hormone (GHRH)-like substance. We examined the ontogeny and pituitary regulation of testicular GHRH-like mRNA (t-GHRH mRNA) and compared this to expression of insulin-like growth factor-I (IGF-I) and IGF-II mRNA in developing testis. t-GHRH mRNA was measured by dot blot hybridization and quantitated using a hypothalamic GHRH cRNA standard. t-GHRH mRNA was not detectable in Northern blots in fetal testis on day 19 of gestation, but was present in low but detectable amounts in testicular dot blots on day 2 of life (0.44 pg/µg total RNA). Levels of the RNA increased beginning on day 21 (1.72 ± 0.23 pg/µg total RNA) and reached adult levels by day 30 (4.96 ± 0.84 pg/µg total RNA). The GHRH species on Northern analysis was about 1750 nucleotides at all ages examined; there was a larger species of about 3350 nucleotides seen on days 65 and 90. There was no correlation between the ontogeny of t-GHRH mRNA and either IGF-I or IGF-II mRNAs, which were maximally expressed in the testes of day 2 animals and decreased with age. To examine the influence of the pituitary gland on t-GHRH mRNA, levels of the mRNA were measured in the testes of hypophysectomized animals and age-matched controls. In animals hypophysectomized on day 21 and killed on day 42 and in animals hypophysectomized on day 42 and killed on day 63, there was marked diminution of t-GHRH mRNA (19 ± 5% and 9 ± 2% of agematched controls, respectively). In contrast, in animals hypophysectomized on day 65 and killed on either day 80 or 90, there was a much smaller difference in levels of t-GHRH mRNA compared to values in control animals (73 ± 20%). This was unlike the effect of hypophysectomy on testicular IGF-I mRNA, where uniform diminution was seen in all three groups. Because GH is important in the regulation of hypothalamic GHRH mRNA, we examined the effects of administration of recombinant human GH on the reinduction of t-GHRH mRNA after hypophysectomy and compared this to the reinduction of IGF-I mRNA. Neither t-GHRH mRNA nor testicular IGF-I mRNA increased in hypophysectomized animals treated with GH.Our results indicate that t-GHRH mRNA is developmentally regulated, and that the hypothalamic-pituitary axis is important in its expression. The influence of the pituitary may be less important in the postpubertal than in the pre- or peripubertal animal. Expression of t-GHRH mRNA is not correlated with expression of either IGF-I or IGF-II in testis. Daily administration of GH alone for 2 weeks is insufficient to alter the diminished expression of this RNA or testicular IGF-I mRNA after prepubertal hypophysectomy. The mechanisms regulating t- GHRH mRNA are complex and may involve pituitary as well as testicular autocrine and paracrine factors.