To evaluate the presence of protein phosphorylation in peripheral blood eosinophils, venous blood was drawn from normal healthy volunteers. Eosinophils were isolated on a Percoll gradient and were incubated with [gamma32P]ATP in the presence of Mg2+. After stopping the reaction, SDS-PAGE was performed and autoradiographs were prepared to determine the incorporation of 32P into proteins. Eosinophils developed at least 24 protein bands below 116.25 kD by SDS-PAGE. In the autoradiographs, one distinct radioactive band was observed with a molecular weight of 65 kD. 32P incorporation into the 65-kD band was dependent on Mg2+ concentration and maximal response was observed at concentrations of 2-6 mM MgCl2. 32P incorporation into the band was dependent on the reaction time and temperature of the reaction system. Acid hydrolysis showed that [32P]phosphate radioactivity in the cells was present primarily as phosphoserine, indicating the presence of 65-kD protein phosphorylation in human peripheral blood eosinophils.