Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gs alpha and Gil alpha. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by approximately 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [32p]p3 (4-azidoanilido)-p1-5'-GTP ([32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha with the extents of nucleotide transfers being 7.6 +/- 0.8% and 17.8 +/- 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, supporting the hypothesis that tubulin contributes to the regulation of neuronal signal transduction.