Phosphor imaging was evaluated for detection, quantitation and resolution of multiphosphorylated protein isoforms separated by two-dimensional gel electrophoresis. A nuclear phosphoprotein, p53, was isolated by immunoprecipitation after biosynthetic labeling with 35S, 32P or 33P in cultured human cells. Of the three radionuclides, 35S was the most sensitive in detection after a 1-week exposure, although shorter exposure times were effective. In dividing cells, 11 35S-labeled isoforms were found, of which 10 were phosphorylated by 33P and 32P. Exposure of phosphonuclides for one half-life showed that 33P radiolabeling produced better resolution among isoforms than 32P but was less sensitive in detection. Volume integration showed phosphorylated isoforms comprised from 1% to 25% of total isoform signal. The relative phosphorylation of each p53 isoform was estimated by normalizing 33P or 32P isoform volumes with the corresponding 35S volume and showed progressive phosphorylation of acidic isoforms. Additionally, phosphor imaging capably detected quantitative changes among individual isoforms after experimental modulation of the isoform pattern by serum deprivation. The described electrophoretic isolation and quantitation procedures should find general application in discerning active and inactive phosphoisoforms for eventual identification.