Aims: In this study we investigated the effects of P2 receptors in the regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in human umbilical vein endothelial cells (HUVEC). Methods: Cytosolic Ca2+ concentration ([Ca2+]i) was measured using fura-2/AM, and MAPK/ ERK phosphorylation using Western blot analysis. Results: ATP, 2-meSATP, UTP and UDP cause a rapid and transitory increase in the phosphorylation of MAPK/ERK. In contrast, negligible response was seen for α,β-meATP, a general P2X receptors agonist. ATP-dependent activation of MAPK/ERK was prevented by pretreatment of HUVEC with pertussis toxin or MEK inhibitor PD98059. In addition, activation of the MAPK/ ERK cascade by ATP was blocked in cells pretreated with wortmannin and LY294002, but not by U73122, BAPTA or a Ca2+-free medium. Furthermore, an inhibition of ATP-dependent MAPK/ERK phosphorylation was observed in HUVEC pretreated with high doses of GF109203X or myristoylated PKC-ζ pseudosubstrate. Similar results were observed when cells were pretreated with the Src tyrosine kinase inhibitor PP2. However, ATP-stimulated MAPK/ERK activation was unaffected in cells pretreated with AG1478 or perillic acid. We also found that ATP stimulates both the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and its translocation to plasma membrane in a time-dependent manner. Conclusion: These observations suggest that the effects mediated by ATP in HUVEC occur via PTX-sensitive G-protein-coupled P2Y receptors through PI3K-dependent mechanisms, in which PDK1 and PKC-ζ are two key molecules within signal cascade leading to MAPK/ERK activation. [ABSTRACT FROM AUTHOR]