The B1 molecule (CD20) is a phosphoprotein expressed only by B-lymphocytes. In this study, analysis of B1 immunoprecipitated from surface iodinated B-cell lines and B-lymphocytes has shown that there are several expressed forms of B1. A predominant species of Mr 33,000 represents 75-80% of the iodinated cell surface B1 and a Mr 35,000 species represents 20-25%. Limited proteinase digestion of these two species generated similar peptide maps demonstrating that the different forms of B1 shared common peptides. Biosynthetic labeling with [35S]methionine revealed that the Mr 35,000 B1 species may actually represent two bands of Mr 34,500 and 36,000. Endoglycosidase digestion studies and metabolic labeling in the presence of tunicamycin indicated that neither the Mr 33,000 or 34,500-36,000 forms of B1 were glycosylated. The Mr 33,000 and 34,500-36,000 forms of B1 were constitutively phosphorylated in B-cell lines. However, exposure of B-cells to PMA resulted in a significant increase in the phosphorylation of the Mr 34,500-36,000 form. Exposure to PMA also resulted in an increase in the amount of Mr 34,500-36,000 protein immunoprecipitated from 35S labeled cells. These results suggest that there are multiple forms of the B1 molecule expressed by B-lymphocytes and that this heterogeneity may result from phosphorylation of the B1 protein.