Proline-rich kinase 2 (Pyk2) is a non-receptor tyrosine kinase belonging to the focal adhesion kinase family. Many stimuli can initiate phosphorylation and activation of Pyk2 but its specific activators and downstream targets are still largely unidentified and little is known of the mechanisms or role of Pyk2 activation in endothelial cells. In human umbilical vein endothelial cells (HUVEC), we show that (1) Pyk2 is phosphorylated on tyrosine residues 402, 580, and 881 in response to stimulation with G-protein-coupled receptor agonists (GPCAs), vascular endothelial growth factor, and the cytokine interleukin-1alpha; (2) HUVEC express mRNA for two isoforms of Pyk2 which do not appear to be regulated transcriptionally by GPCAs, growth factors, or cytokines; and (3) Pyk2 is localised to the cytosol and associates through its C-terminus with the cytoskeletal protein paxillin and the adapter molecule p130Cas in phosphorylation-independent interactions. These results demonstrate that Pyk2 is rapidly activated and associates with structural and adapter proteins suggesting that it is an important kinase involved in mediating acute responses in endothelium.