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To investigate the mechanisms of vascular uptake of prorenin and renin and to explore the possibility of vascular activation of prorenin.Human umbilical vein endothelial cells (HUVECs) cultured in a chemically defined medium were incubated with recombinant human prorenin or renin in the presence or absence of putative inhibitors of renin internalization. Cell surface-bound and internalized prorenin or renin were separated by the acid-wash method and were quantified by enzyme-kinetic assays. The activation of prorenin was also monitored by a direct immunoradiometric assay (IRMA) with use of a monoclonal antibody directed against the -p24-Arg to −1 p-Arg C-terminal propeptide sequence of prorenin.Prorenin and renin were internalized at 37°C in a dose-dependent manner; with 1000 μU prorenin/ml medium, the quantity of cell-associated prorenin after 3 h of incubation was 9.3 ± 1.0 μU/4 × 105 cells, and with 75 000 μU/ml medium it was 670 ± 75 μU/4 × 105 cells (mean ± SD; n = 5). Results for renin were similar. Prorenin that had been treated with endoglycosidase H to remove N-linked oligosaccharides was not internalized. Addition of mannose 6-phosphate (M-6-P) to the medium caused a dose-dependent inhibition of renin and prorenin internalization. Fifty per cent inhibition was observed at 70 μmol/M-6-P, whereas mannose 1-phosphate, glucose 6-phosphate and α-methylmannoside at this concentration had no effect Ammonium chloride (50 mmol/l) and monensin (10 μmol/l) also inhibited internalization. Prorenin was activated by HUVECs, and cell-activated prorenin was only found in the internalized fraction, whereas the surface-bound prorenin remained inactive. Thus, it appears that the activation of prorenin took place at the time of its internalization or thereafter. The results of the prorenin IRMA indicated that activation was associated with proteolytic cleavage of the propeptide.Our findings provide evidence for M-6-P receptor-dependent endocytosis of (pro)renin and proteolytic prorenin activation by vascular endothelial cells.