The diversity of monocyte chemotactic protein (MCP)3 target cell types, as well as the capacity of MCP3 to desensitize leukocyte responses to other CC chemokines, suggested that MCP3 may interact with multiple CC chemokine receptors. The purpose of this study is to establish how MCP3 binds and activates monocytes and neutrophils. We show that human monocytes exhibit high-affinity binding for 125I-MCP3 with an estimated Kd of 1-3 nM and about 10,000 binding sites/cell. The binding of 125I-MCP3 to monocytes was progressively less well competed by CC chemokines macrophage inflammatory protein (MIP)1 alpha (Kd = 5-10 nM), RANTES (Kd = 5-10 nM), MCP1 (monocyte chemoattractant and activating factor, or MCAF) (Kd = 60 nM) and MIP1 beta (Kd > 100 nM). On the other hand, unlabeled MCP3 displaced the binding of radiolabeled MIP1 alpha, RANTES, MCP1 and MIP1 beta as effectively as the isologous CC chemokines. In agreement with the binding data, pretreatment of monocytes with MCP3 completely desensitized the calcium flux in response to MIP1 alpha and RANTES. However, MIP1 alpha and RANTES failed to desensitize the response of monocytes to MCP3. MCP3 and MCP1 partially desensitized each other's effects on monocytes. These binding and cross-desensitization results suggest that MCP3 binds and signals through other binding sites in addition to those shared with MIP1 alpha, RANTES and MCP1. The unidirectional competition for MIP1 beta binding and signaling by MCP3 suggests the existence of an as-yet unidentified site for MCP3 shared with MIP1 beta. The existence of another unique binding site(s) for MCP3 was further shown by the failure of any of the other CC chemokines to compete effectively for MCP3 binding on neutrophils. MCP3 in our study was also the only human CC chemokine that consistently chemoattracted neutrophils. These results suggest that MCP3 is a ligand that can bind and activate a broad range of target cells through receptors shared by other CC chemokines as well as its own receptor.