The bactericidal and opsonic properties of serum obtained from a strain of guinea pigs totally lacking in hemolytically active or antigenically detectable C4 were studied and compared to serum from normal guinea pigs and those heterozygous for C4 deficiency. Bactericidal activity against two strains of E. coli was present in C4-deficiency serum; however, the latent period was prolonged and the rate was slower when compared to that in normal or heterozygous sera. C4-deficient serum bactericidal activity could be restored to normal by mixing with normal serum or by adding partially purified C4 from human serum. Treatment of both C4-deficient and normal serum with heat (56°C for 30 min), 0.04 M EDTA, and zymosan abolished bactericidal activity, whereas 0.03 M hydrazine treatment impaired but did not completely destroy the activity of both sera. C4-deficient serum promoted phagocytosis of S. aureus, type 25 pneumococci, E. coli and C. albicans but kinetic studies employing 14C labeled bacteria indicated that ingestion rates were slower than in normal or heterozygous serum. Prior incubation of pneumococci in serum established that opsonization was the rate-limiting step in phagocytosis of these organisms and that this was significantly diminished from normal in C4-deficient serum. Normal opsonization could be restored by the addition of as few as 10 units of partially purified guinea pig C4. Treatment with heat, EDTA and zymosan abolished opsonization in both normal and C4-deficient sera. Hydrazine treatment diminished the opsonic activity of normal serum against pneumococci but did not significantly affect C4-deficient serum pneumococcal opsonization. These observations are consistent with the operation of an alternate pathway for activation of late complement components necessary for bacterial opsonization and killing by serum, but suggest that participation of C4 presumably through the classic C1,4,2,3–9 sequence might be necessary for maximal rates of activity. In addition, the results of studies using the various complement inhibitors offer support for the participation of heat labile factors, divalent cations and zymosan and hydrazine inhibitable components in the alternate pathway. Finally, both normal and C4-deficient guinea pigs were found to be highly resistant to intraperitoneal administration of high doses of type 25 pneumococci or E. coli used in the in vitro studies. Limited studies of the effects of intravenous challenge with pneumococci likewise revealed no striking impairment in the survival of the C4-deficient animals, suggesting that by utilization of the alternate pathway the host defenses are adequately maintained in vivo.