We have previously shown that platelet factor 4 (PF4), a platelet-specific CXC chemokine, can directly and specifically inhibit human megakaryocyte colony formation. We therefore hypothesized that PF4 might function as a negative autocrine regulator of megakaryocytopoiesis. Herein we present additional studies characterizing the inhibitory effect of CXC chemokines on human megakaryocyte development. We first corroborated our initial studies by showing that recombinant human (rH) PF4, like the native protein, inhibited megakaryocytopoiesis. We then examined the inhibitory properties of other CXC family members. Neutrophil activating peptide-2 (NAP-2), a naturally occurring N-terminally cleaved beta TG peptide, was found to inhibit megakaryocytopoiesis with two to three orders of magnitude greater potency than PF4. Structure function studies showed that an N-terminal mutation, which eliminated NAP-22s neutrophil activating properties (NAP-2E2-->A), also abrogated its ability to inhibit megakaryocyte development. Further investigations of this type demonstrated that a chimeric PF4 protein (AELR/PF4) in which PF42s N- terminus was replaced with the first four amino acids of NAP-2 was also a potent inhibitor of megakaryocytopoiesis. Interleukin (IL)-8, another CXC chemokine, and three CC chemokines (macrophage inhibitory protein-1 alpha [MIP-1 alpha], MIP-1 beta, and C10) also specifically inhibited megakaryocyte colony formation at NAP-2 equivalent doses. CXC and CC chemokine inhibition was additive suggesting that the effects might be mediated through a common pathway. The inhibitory effects of NAP-2 and MIP-1 alpha could not be overcome by adding physiologically relevant amounts of recombinant human megakaryocyte growth and development factor (MGDR) (50 ng/mL) to the cultures. Using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) based analyses, we documented mRNA expression of IL-8 receptor isoforms alpha and beta in total platelet RNA and in normal human megakaryocytes, respectively. Based on these results, we hypothesize that chemokines play a physiologic role in regulating megakaryocytopoiesis. Because chemokines are elaborated by ancillary marrow cells, both autocrine and paracrine growth control is suggested, the effects of which might be exerted, in part, through alpha and beta IL-8 receptors.