Abstract An ectoprotein kinase activity has been identified on intact rabbit peritoneal polymorphonuclear leucocytes and the time course of phosphate incorporation into proteins has been followed at different ATP levels. Saturation is reached at around 3 mM ATP and the activity is inhibited by p-chloromercuribenzoate. The possibility that the observed protein phosphorylation arises through the action of a membrane ATPase liberating phosphate for transfer into the cell, incorporation into ATP and its utilisation by endogenous kinases, has been excluded by studying both enzymes concomitantly and measuring the rate of [ 32P]orthophosphate uptake. Lactate dehydrogenase measurements in the extracellular media also exclude the possiblity of kinase liberation from lysed cells. Moreover, the pattern of 32P-labelling of polypeptides when intact cells are exposed to [ 32P]ATP is quite different from that when homogenates are incubated with [ 32P]ATP or intact cells with [ 32P]-orthophosphate. We have been unable to demonstrate any cAMP dependency for this ectokinase activity.