C3b and iC3b, opsonic fragments of C3, interact with specific receptors on phagocytic cells. After bacterial opsonization, C3 fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blotting, and immunodetection. For bacteria opsonized in 50% pooled human serum (PHS), C3 deposition and cleavageto iC3b occurred rapidly. C3b, iC3b, and C3d made up 17%, 64%, and 19%, respectively, of the C3 on Staphylococcus aureus and 53%, 44%, and 2%, respectively, on Escherichia coli. Residual C3b was refractory to factor I cleavage,an occurrence enabling alternative pathway activation to continue. C3 deposited was quantitated by enzyme-linked immunosorbent assay; with 50% PHS, >50% and 90% of total C3 deposition occurred within 5 and 10 min, respectively. With a lower percentage of PHS, maximal deposition required up to 60 min and was not achieved in <10% PHS. Ester-bound fragments represented 34% and 82% of covalently bound C3 on S. aureus and E. coli, respectively.