Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.