We have investigated the ontogeny of immunoreactive β-endorphin (i-βE) in the testes of rats from 5 to 150 days of age. i-βE was measured by RIA in acid extracts of decapsulated testes and characterized by gel filtration chromatography. Significant age-related differences in both the levels and type of i-βE were observed. Total levels of i-βE in the testes were very low and barely detectable from 5-20 days of age, but rose sharply in parallel with testes weights from 20-60 days of age; thereafter, no significant changes in i-βE were found through 150 days of age. Concentrations of i-βE, expressed in pmol/g testis, fell precipitously between days 5 and 10 and remained relatively constant from 10-150 days. Most of the i-βE at 5 and 15 days chromatographed like authentic β-endorphin. However, with the onset of puberty (30-35 days) and during sexual maturation, much of the total i-βE chromatographed like its' precursor β-lipotropin (βLPH). Hypophysectomy decreased the weight and total i-βE levels of the testes to the same extent without altering the concentrations of i-βE or the chromatographic pattern of i-βE. These results indicate that βE-like and βLPH-like peptides are present in the rat testis and that age-related changes in both the levels and type of i-βE correlate with various structural and functional aspects of testicular development.