Various data in the literature suggest that progenitor cells of monocytes and B cells are closely related. In this study we have investigated this notion by using two-dimensional gel electrophoresis patterns of total cellular phosphoproteins to assess the similarity and thus the close relationships between erythroid, granulocyte, monocyte, and B-lymphocyte cell lines that typify immature cells of these lineages. In previous studies, six proteins had been identified whose constitutive phosphorylation correlated with the capacity of HL60 variant cell lines to differentiate towards either neutrophils (four proteins) or monocytes (two proteins). The presence or absence of five of these phosphoproteins in autoradiographs obtained for the pre-B-cell lines Nalm6 and SMSB showed that the pre-B-cell lines most closely resembled lines able to differentiate towards monocytes (HL6015-12, HL60M2, U937, and ML-1) as opposed to lines restricted to neutrophil (HL60Ast3) or erythroid differentiation (K562). The K562 phosphoprotein pattern resembled that obtained for HL60Ast3. Progressive changes in the constitutive phosphorylation of the five proteins were observed suggesting that cells that have acquired the potential for either neutrophil, monocyte, or B-cell differentiation have initially diversified in a linear progressive manner. This observation supports a model for hemopoiesis that suggests that, during progenitor cell development, differentiation potentials are expressed individually in the above order. Two additional phosphoprotein spots were found to be restricted to the pre-B-cell lines. These phosphoproteins, together with those that change their intensity in autoradiographs of erythroid, myeloid, and B-cell lines, suggest that protein phosphorylation plays an important role during cell diversification.