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Normal human serum absorbed at 0 degrees C with pneumococcal serotype 1, 12, or 25 lost the ability to support polymorphonuclear leukocyte intracellular killing of some pneumococcal serotypes even if immunoglobulin was provided. The absorbed serum contained no organisms but had residual polysaccharide when measured by counterimmunoelectrophoresis against type-specific antisera. The influence of pneumococcal polysaccharide (PPS) on serum support of intracellular polymorphonuclear leukocyte killing was evaluated. Normal human serum was mixed with PPS serotype 1, 12, or 25 at 0 degrees C for up to 120 min and then used as the opsonic source in standard phagocytic killing assays with serotype 1, 12, or 25. Immediately after mixing, each serum combined with PPS serotype 1, 12, or 25 supported killing of all serotypes tested. With increasing time at 0 degrees C with serotype 1 PPS, serum exhibited a marked progressive decline in killing of serotype 25, a similar but less marked impairment in killing of serotype 12, and essentially no impairment for serotype 1. Serum treated with type 25 PPS did not support killing of type 25, but allowed normal killing of types 1 and 12. Incubation with type 12 PPS impaired opsonization of types 12 and 25, but not 1. Addition of PPS-specific antisera did not restore killing. Residual serum hemolytic activity of classic and alternative complement pathways was not reduced below opsonizing levels after 120 min at 0 degrees C. Furthermore, PPS treatment of normal human serum did not alter the attachment or ingestion of 14C-labeled pneumococci by polymorphonuclear leukocytes. Some serotypes of PPS reacted slowly with serum at 0 degrees C, diminishing its ability to support intracellular killing of pneumococci despite serum hemolytic complement activity. Phagocytosis was not inhibited. Specific antibody did not overcome inactivation. Different requirements for the inactivated factor among serotypes may be a characteristic related to organism virulence.