Extracellular protein kinase activity is demonstrated in intact cultured porcine aortic endothelial cells and is characterised. When cells were incubated with [gamma-32P]ATP (1 microM) a major cell surface protein, corresponding to 115 kDa, and at least four serum proteins (19, 21, 55 and 126 kDa) became phosphorylated. Protein kinase activity is released by intact endothelial cells, which is not due to cell damage, as judged by various cell viability parameters (e.g., release of marker enzymes, trypan blue exclusion). The activity of the protein kinase released amounted to 170 fmol/min per mg endothelial cell protein with phosvitin as substrate, which represents 9% of the total cellular phosvitin protein kinase activity. Repetitive incubation of endothelial cells substantially decreased phosvitin-kinase release. Exo-protein kinase is not influenced by cAMP and cGMP but is effectively inhibited by heparin (EC50, 0.3 microgram/ml). The findings clearly demonstrate: (1) exo-protein kinase is released by intact porcine aortic endothelial cells; (2) substrates of this enzyme are endothelial surface proteins and serum proteins.