The recently discovered CC chemokine, regakine-1, is constitutively present in bovine serum and synergizes with the CXC chemokine interleukin-8 (IL-8) to chemoattract neutrophils. Here we show that regakine-1 cooperates with the CXC chemokine receptor 2 ligand neutrophil activating protein-2 (NAP-2) and the anaphylatoxin C5a, two other mediators of inflammation present in the circulation. Neutrophil chemotaxis was 3-fold enhanced when regakine-1 (100 ng/ml) and C5a (30 ng/ml) were combined at concentrations present in bovine or human plasma, respectively. This synergy was also observed when neutrophils were preincubated with regakine-1. Plasma chemokines such as NAP-2, beta-thromboglobulin, and hemofiltrate CC-chemokine-1 did not affect C5a chemotactic activity. The capability of regakine-1 to synergize with C5a, NAP-2, or N-formyl-methionyl-leucyl-phenylalanine (fMLP) was not observed for monocyte chemotactic protein-3 (MCP-3), another CC chemokine that weakly chemoattracts neutrophils. Regakine-1 also failed to cooperate with MCP-3 and macrophage inflammatory protein-1alpha in neutrophil chemotaxis. The receptor of regakine-1 is not known yet. Competition with labeled fMLP or C5a for binding to neutrophils or receptor transfected cell lines demonstrated that regakine-1 did not alter receptor recognition. The protein kinase inhibitors 2'-amino-3'-methoxyflavone (PD98059), wortmannin and staurosporin had no effect on the synergy between C5a and regakine-1. Although NH2-terminal truncation affects the chemotactic potency of most chemokines, it did not affect the synergistic capacity of regakine-1 with C5a on neutrophils. These findings indicate that the constitutive plasma chemokine regakine-1 is a stable enhancer of the inflammatory response and that its blockade might be beneficial in acute and systemic inflammatory disorders.