The testis contains many peptides originally described as originating in the central nervous system. The physiological function of these factors in the testis is generally unknown. We previously reported that the rat testis contains both a peptide with GH-releasing hormone-like immunoactivity (tGHRH-LI) and a mRNA species that cross-hybridizes with a hypothalamic cDNA for rat GHRH (rGHRH). The current study was designed to further characterize tGHRH-LI by determining its location within rat testis, and to evaluate whether tGHRHLI and hypothalamic GHRH share similar biological and electrophoretic properties.Partially purified tGHRH is capable of stimulating GH secretion from cultured anterior pituitary cells in a dose-dependent manner. Testicular GHRH and rGHRH have different HPLC retention times and significantly different electrophoretic properties by Western gel analysis. The estimated size of tGHRHLI is approximately 3.7 times that of synthetic rGHRH. Using immunohistochemistry, tGHRH-LI is localized to mature sperm forms in rat testis. We conclude that rat tGHRH-LI and rGHRH share some structural and functional properties and are probably related peptides. However, the difference in electrophoretic mobility and HPLC retention time indicates that they are not identical. The presence of tGHRH-LI in rat sperm, within the confines of the blood-testis barrier, which is generally impermeable to peptides, leads us to speculate that tGHRH serves a paracrine or autocrine role in testicular physiology.