The binding and internalization of recombinant human renin and prorenin (2500 μU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37°C and 4°C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37°C, after incubation at 4°C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37°C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor–dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.