We have analyzed the detailed structure and cytoplasmic distribution of cytoplasmic microtubule-associated proteins. The procedure used to identify these proteins, based on preparation of detergent-extracted cytoskeletons, permits separation of fractions containing assembled and unassembled microtubule proteins. We show that two of these proteins, 69 and 80 kd, are closely related to one another and that each protein is present as a set of structurally related polypeptides with differing isoelectric points. In both neuroblastoma and pheochromocytoma cells, several of the isoelectric variants are greatly enriched in the fraction containing assembled microtubule components. Their differential distribution is correlated with phosphorylation at novel sites on the protein. These results support the possibility that covalent modification of a cytoskeletal component may specify its functional state.