Trypan blue completely inhibited attachment of human polymorphonuclear leukocytes (PMN) to Sepharose beads coated with C3 and to sheep erythrocytes coated with IgM plus C3, but it did not inhibit attachment to erythrocytes coated with IgG. These results suggested that trypan blue inhibited C-mediated attachment to PMN membranes. Corroborative studies were performed with a strain of Staphylococcus aureus that requires C but not antibody, for opsonization and that activates the alternative pathway. Trypan blue was not toxic to PMN or bacteria, did not interfere with immunoglobulin or C interactions, and did not affect attachment of opsonins to bacteria. However, the dye impaired PMN killing of S. aureus in normal nonimmune serum by inhibiting bacterial attachment to and ingestion by PMN. Further evidence that the inhibition was at the C3 receptor level came from the observations that, 1) once staphylococci were attached to PMN at either 37°C or 0°C, addition of trypan blue did not inhibit killing; and 2) trypan blue inhibited killing of bacteria opsonized with serum sufficient in C but previously absorbed at 0°C with the same strain of organism to deplete specific antibody. Further studies with this agent may elucidate the roles of opsonic receptors on human phagocytes.