Monocyte chemotactic protein (MCP)-2 is a member of the C-C chemokine subfamily, which shares more than 60% sequence homology with MCP-1 and MCP-3 and about 30% homology with macrophage inflammatory protein (MIP)-1alpha, regulated on activation of normal T cell expressed (RANTES), and MIP-1beta. Despite this considerable sequence homology to other C-C chemokines, MCP-2 appears to have unique functional properties in comparison with other C-C chemokines such as MCP-1 and MCP-3. Although evidence obtained from studies on leukocytes suggested that MCP-2 may share the receptors with these C-C chemokines, the actual functional receptors for MCP-2 have not yet been identified. In this study, by using radioiodinated MCP-2, we identified high affinity binding sites for MCP-2 on human peripheral blood monocytes. The MCP-2 binding was competed for by MCP-1 and MCP-3, but less well by MIP-1alpha or RANTES. In experiments using cells transfected with C-C chemokine receptors, 125I-MCP-2 bound to human embryonic kidney 293 cells transfected with CCR1 or CCR2B, known to also bind MIP-1alpha/RANTES and MCP-1, respectively, but both shared by MCP-3. The binding of 125I-MCP-2 to these receptor-transfected cells was displaced completely by chemokines that bind to these receptors. Both CCR1- and CCR2B-transfected 293 cells showed significant migration in response to MCP-2, in addition to responding to other specific chemokines. These results clearly demonstrate that MCP-2, unlike MCP-1, uses both CCR1 as well as CCR2B as its functional receptors, and this accounts for the unique activities of MCP-2.