The gene encoding the proenkephalin precursor is expressed in both progenitor spermatogenic cells and somatic cells in the rat testis. The 1750-nucleotide (nt) germ cell-specific proenkephalin mRNA is the predominant form detected in the adult rat. The 1400-nt somatic cell form has previously been shown to be expressed in cultured Sertoli cells derived from sexually immature rats, and FSH treatment increases expression of the proenkephalin gene in that experimental system. In the present report we demonstrate that the 1400-nt proenkephalin transcript is present in a freshly isolated, enriched preparation of adult rat Leydig cells. This gene continues to be expressed when the Leydig cells are maintained in primary culture. Treatment of cultured Leydig cells with hCG or a cAMP analog leads to a rapid increase in proenkephalin-mRNA levels. The expression of the proenkephalin gene throughout rat development in the two major somatic cell types of the testis further suggests that proenkephalin-derived peptides play an important role in modulating testicular function. The gene encoding another of the opioid peptide precursors, POMC, has previously been reported to be expressed in rat Leydig cells. We were unable to detect POMC transcripts in cultured Leydig cells. POMC mRNA is abundant in a germ cell-enriched cell fraction.