The phosphorylation sites of spectrin dimer were characterized before and after incubation of intact human red cells with [32P]orthophosphate. Phosphate measurements of unlabeled spectrin dimer show it contains 4.0 +/- 0.3 covalent protein phosphates, all located on band 2. Quantitation of the number of exchangeable phosphorylation sites in erythrocytes labeled with [32P]orthophosphate yields 3.9 +/- 0.3 phosphates/spectrin dimer, indicating that all four spectrin phosphorylation sites are metabolically active. Tryptic and chymotryptic peptide mapping reveals that the dimer contains three unique 32P-labeled tryptic peptides of approximately 4,600 (A1), 3,500 (A2), and 2,400 (B) daltons. Peptide A1 contains both phosphoserine and phosphothreonine while peptides A2 and B possess only phosphoserine. Peptides A1 and A2 remain associated after trypsinization of spectrin dimer and are only separable in detergents. All three 32P-labeled tryptic peptides are contained within a 20,000 dalton cyanogen bromide fragment which is within a 60,000 dalton staphylococcal protease phosphopeptide. All these fragments are found within a 90,000 dalton nitrothiocyanobenzoic acid phosphopeptide. The purified 20,000 dalton fragment contains no homoserine or homoserine lactone and is the COOH-terminal cyanogen bromide peptide of band 2. The isolated tryptic peptide B possesses no lysine or arginine and is presumably the COOH-terminal tryptic peptide of band 2 and the most distal phosphopeptide. Thus, the four phosphorylation sites of spectrin dimer are clustered at the extreme COOH-terminal end of band 2.