Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.