We have developed a simple method for phosphoamino acid analysis of 32Plabeled phosphoproteins using anion-exchange high-performance liquid chromatography (HPLC). Phosphoproteins undergo partial acid hydrolysis and the resulting hydrolysate is injected directly onto a column. The sample is then isocratically eluted from the column by 35 mM phosphoric acid at pH 3.0 with collected fractions analyzed by Cerenkov counting. Phosphoamino acid identification is accomplished by the comparison of the retention times of 32P-labeled peaks to retention times of phosphoamino acid standards which had been monitored at 206 nm. This method has greater sensitivity and is more reliable than cellulose thin-layer electrophoresis and the results obtained by high-efficiency Cerenkov counting can be evaluated immediately, instead of waiting days or weeks for autoradiographic development of cellulose plates. This HPLC protocol is an improvement over other published HPLC protocols in that there is no need for pre-or post-column derivatization and the free [32P]phosphate elutes long after the phosphoamino acids. Thus sensitivity is increased as there is no interference from the free phosphate. Selection of an HPLC anionexchange column is critical for this separation. Only two of the four columns that we tested performed well. We present data from several phosphoproteins including calcium-calmodulin dependent protein kinase, the p-subunit of the insulin receptor, and phosphorylated calmodulin to demonstrate the utility of this procedure.