The nature of the opsonic factors in nonimmune human serum for six blood culture isolates of Pseudomonas aeruginosa was investigated by measuring uptake of [3H] adenine-labeled bacteria by human PMNs. Normal human serum, C2- and C4-deficient sera, zymosan-treated serum, and immunoglobulin-deficient sera were used as opsonic sources. Heat inactivation of each of these serum sources markedly reduced its opsonic capacity for all Pseudomonas strains, suggesting that the serum C system was essential for opsonization. Five strains were opsonized in the absence of the classical C pathway; however, kinetic studies revealed that opsonization proceeded at a faster rate when the classical pathway was present. In spite of markedly reduced factor B and C3 levels, zymosan-treated serum retained significant opsonic activity for one of the strains tested. Four strains were poorly opsonized by immunoglobulin-deficient serum, and C activation by these strains appeared to depend upon the presence of antibodies. Two strains, however, were effectively opsonized in a relative absence of antibodies. Thus, in the nonimmune state, phagocytosis of P. aeruginosa is mediated primarily via the C system, and antibodies appear to play a role in the opsonization of some but perhaps not all Pseudomonas strains.