Previous studies have shown that the interaction of pneumococci and the heat labile opsonin (HLO) system results in the cleavage of C3 and the fixation of C3b on the surface of the pneumococcus. This process is not a direct interaction, but requires the participation of other factors present in normal guinea pig serum (GPS). The present studies concern the identity of these factors. One component of the HLO system is guinea pig γ2 immunoglobulin. It is bound to the pneumococcus during opsonization and can be demonstrated to participate functionally in the interaction of the pneumococcus and the HLO system. Using an in vitro phagocytic test, we also found that C4 deficient GPS is able to support the phagocytosis of pneumococci by polymorphonuclear leukocytes as well as normal GPS. Also, when pneumococci are incubated in C4 deficient GPS, they interact with the HLO system to consume C3 as well as they do when incubated in normal GPS. Therefore, C4 is not required for the opsonization of pneumococci by the HLO system and the cleavage and fixation of C3 probably proceeds via the alternate complement pathway.