Hormone-responsive peptides play a vital role in development and regulation of testicular function. The preoptic regulatory factors, porf-1 and porf-2, were originally discovered in the rat brain, but are also expressed in the rat and human testis. In the brain expression is age-related, hormone-responsive, region- specific, and gender-related, suggesting that porf-1 and porf-2 are involved in gender-specific brain development and function. Tissue-specific porf-1 and porf-2 mRNAs are also found in the testis and hypophysectomy may alter testicular porf-2 expression. It was thus of interest to further examine porf-1 and porf-2 expression in the testis to evaluate their potential as hormone-responsive peptides that regulate testicular development and function. Testicular expression of both porf-1 and -2 was analyzed as a function of maturational stage, aging and hypophysectomy by the solution hybridization/nuclease protection assay, and cellular location determined by in situ hybridization histochemistry. Expression was quantitatively compared in normal male rats at 15, 30 and 60 d (n = 4) and at 2, 6, 12, and 24 mo of age (n = 5). During development porf-1 is expressed at a constant level at 15, 30 and 60 d, then declines significantly with advancing age; levels at 24 mo are only 20% of those seen at 2 mo (p < 0.05). In contrast, porf-2 expression is highest at 15 d of age and steadily declines at 30 and 60 d, plateaus in the mature adult (6 and 12 mo), then exhibits an additional significant decline in the aged 24 mo animals (6 vs 24 mo, p < 0.05). Hypophysectomy of young adult rats at day 42 results in increased testicular expression 12 d later of both porf-1 (p < 0.05) and porf-2(p < 0.005) compared to intact 54-d-old rats (n = 5). In situ hybridization histochemistry confirms that both porf-1 and porf-2 are expressed in the mature testis at 60 d of age. Porf-2 mRNA is localized to immature germ cells including spermatogonia and primary spermatocytes. Porf-1 mRNA is associated with mature sperm and at low levels in the Sertoli cell cytoplasm surrounding spermatocytes. These data suggest that porf-2 is a pituitary hormone-responsive factor in the developing testis and that both porf-1 and porf-2 have cell-type specific functions in the germ cell compartment of the mature testis