Human erythrocyte band 3 is a major substrate of two red blood cell protein kinases, casein kinase I and p72syk protein tyrosine kinase. Although the phosphorylation sites and physiologic consequences of p72syk phosphorylation have been characterized, little is known regarding casein kinase I phosphorylation. In this report, we identify the major phosphorylation site of casein kinase I. Using isolated components, casein kinase I was found to phosphorylate the cytoplasmic domain of band 3 (CDB3), primarily on Thr residues. Classical peptide mapping narrowed the major phosphorylation site to a peptide encompassing residues 24-91. Computer-assisted evaluation of this sequence not only showed two consensus casein kinase I phosphorylation sites, but also provided information on how to proteolytically separate and isolate the candidate sites. Following the suggested protocols, a heptapeptide containing the major phosphorylation site was isolated, subjected to amino acid sequencing, and found to be phosphorylated on Thr 42. A minor phosphorylation site was similarly identified as Ser 303. Because Thr 42 is situated near the binding sites on CDB3 of ankyrin, protein 4.1, protein 4.2, and the glycolytic enzymes, phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or function.