The pharmacokinetics, tissue distribution, cell localization, and penetration into tumor xenografts of recombinant [35S]methionine-labeled human alpha interferon (HuIFN-alpha) and murine alpha interferon (MuIFN-alpha) were examined in mice. Both interferons (IFNs) were removed from the blood in a rapid biphasic manner; HuIFN-alpha was cleared faster than MuIFN-alpha. Tissues were analyzed for radioactivity and over 90% of the IFNs was accounted for. The IFNs were detected predominantly in liver, kidney, gastrointestinal tract, pancreas, spleen, and lung. The levels of MuIFN-alpha compared with HuIFN-alpha were greater in the liver, spleen, and lung and less in the kidney, pancreas, and gastrointestinal tract. Heart, brain, testes, thymus, lymph nodes, fat, skin, and skeletal muscle contained much lower but measurable levels of both IFNs. There was penetration of HuIFN-alpha into tumor xenografts. The pharmacokinetics of IFN-alpha were independent of the strain of mouse, BALB/c or CBA, immune deprivation, or the presence of a tumor xenograft. Autoradiography of tissue sections from mice given injections of HuIFN-alpha or MuIFN-alpha indicated focal radioactivity in proximal convoluted tubules in the kidney and diffuse radioactivity in the liver, gastrointestinal tract, and pancrease. MuIFN-alpha, but not HuIFN-alpha, showed intense localization in cells in hepatic sinusoids, marginal zones in the spleen, and pulmonary alveolar walls, suggesting uptake by cells of the monocyte/macrophage lineage in these sites. The study shows the utility of biosynthetic labeling for pharmacokinetic studies of cytokines, clear differences in tissue distribution of IFN-alpha according to its species of origin, and targeting of homologous IFN-alpha to cells of the monocytic lineage.