Alpha 1-proteinase inhibitor, metabolically labeled with 35S, was detected in concentrated culture medium of rat alveolar macrophages by immunoelectrophoresis autoradiography. The metabolically labeled macrophage alpha 1-proteinase inhibitor had the same electrophoretic mobility as did pure rat serum alpha 1-proteinase inhibitor. Moreover, monospecific antiserum to rat serum alpha 1-proteinase inhibitor precipitated a 35S-labeled protein from macrophage culture supernates, and this protein was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to have a molecular weight close to that of rat serum alpha 1-proteinase inhibitor. Cycloheximide (an inhibitor of protein synthesis) at a concentration of 0.50 micrograms/ml, inhibited secretion of labeled inhibitor. This, cultured rat alveolar macrophages secrete detectable concentrations of a proteinase inhibitor believed to play a major role in lung defense.