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The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.