Prostate-specific antigen (PSA) is a chymotrypsin-like serine protease exclusively produced by the prostate epithelium, and abundant in seminal fluid. In serum, PSA is predominantly complexed to a liver-derived serine protease inhibitor, alpha-1-antichymotrypsin (ACT). A higher proportion of serum PSA is complexed to ACT in prostate cancer than in benign prostate hyperplasia. Since the molecular basis for this is unclear, we have investigated whether or not ACT may be produced in the prostate gland. Immunocytochemistry, using either monoclonal or polyclonal IgGs, demonstrated specific immunostaining for ACT in normal PSA-containing prostate epithelium. Production of ACT in the normal PSA-producing prostate epithelium was demonstrated by means of nonradioactive in situ hybridization using 30-mer anti-sense DNA probes for ACT and for PSA. The ACT and PSA coding transcripts, as detected by in situ hybridization, were distributed perinuclearly, in contrast to the specific immunostaining for ACT and PSA which was most intense in the apical portion of the secretory cells. The results strongly suggest local production and release of ACT by the normal prostate epithelium that may be important for interaction between PSA and ACT in extracellular compartments.