Human polymorphonuclear leukocytes (PMNL) display the ability to bind particles coated with C3b or C3bi in the absence of immunoglobulin. Using highly purified, defined fragments of C3, we have extended the eivdence which indicates that these cells possess two distinct C3 receptors which are specific for their respective ligands but not for C3d. PMNL rosettes with erythrocytes (E) bearing only C3b (EC3b) were inhibited by soluble C3b but not native C3, C3bi, C3c, or C3d. EC3bi rosettes were inhibited by soluble C3bi but not native C3, C3b, C3c, or C3d. Engagement of either C3 receptor elicited comparable biological responses from PMNL. Particle bound C3b or C3bi stimulated the production of active oxygen species by PMNL as detected using a chemiluminescence (CL) assay. CL responses occurred in the total absence of immunoglobulin and were inhibited by sodium azide or superoxide dismutase but not by catalase. Particle bound C4b also elicitied a CL response from PMNL. Engagement of multiple C3 receptors was necessary to stimulate the PMNL. Although soluble, monomeric C3b had no effect on PMNL, polymeric forms of C3b induced a CL response. Using a 51Cr release assay, the antibody-dependent, PMNL-mediated killing of Raji cells was enhanced three- to fourfold when targets carried C3 fragments on their surface. Antibody was not required to effect phagocytosis of C3 fragment coated Escherichia coli. Fluoresceinlabeled E. coli 04 were preopsonized by incubation with a mixture of the six purified alternative pathway proteins at their respective physiological concentrations which were totally devoid of immunoglobulin. Incubation of the treated E. coli with purified PMNL led to ingestion of the bacteria as assessed by a combination of phase contrast and fluorescent light microscopy and scanning and transmission electron microscopy. Using radiolabeled E. coli 075, either C3b or C3bi was found to be sufficient to induce antibody-independent ingestion of the bacteria by PMNL. These studies thus comprise the first description of PMNL reactions which depend upon or are enhanced by C3bi. By utilizing well-defined and purified C3 fragments, these studies also clearly illustrate the participation of cell bound C3 fragments in promoting both antibody-dependent as well as antibody-independent cytotoxic reactions mediated by PMNL.