A somewhat simplified modification of a previously described method for the measurement of red cell membrane phosphorylation by ATP has been devised. Phosphorylation of membranes was linear with time for only 5- 10 min, and linearity with membrane concentration was observed only when assays were limited to short incubation times. Protein kinase activity of hereditary spherocytosis (HS) membranes was found to be normal. However, the average phosphorylation after 60 min incubation was less in HS membranes than in normal membranes. Findings similar to those in HS membranes were observed in sickle cell disease. The Km of red cell protein kinase for ATP is approximately 10(-5) M. Membrane phosphate binding sites are not saturated in either HS or normal membranes after 1 hr incubation with ATP. Approximately 27% of phosphorylating activity is lost after 1 hr incubation at 37 degrees C. GTP is a very inefficient phosphate donor. Under the conditions of measurement employed, the enzyme is slightly stimulated by 1 muM cAMP, but is not stimulated by 1 muM cGMP. Dephosphorylation of red cell membranes after labeling occurs at a similar rate in HS as in normal membranes. Although a mild abnormally in membrane phosphorylation is observed in HS, this could not be demonstrated to be due to a decrease in protein kinase activity or in alterations of its kinetic properties. The abnormally seen is not specific for HS.