Abstract: The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2‐month‐old and 24‐month‐old rats. The hydrolysis‐resistant GTP analogue 5′‐guanylylimidodiphosphate (GppNHp)‐dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24‐month‐old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (∼28%) in 2‐month‐old rats. Tubulin‐GppNHp‐dependent inhibition of adenylyl cyclase in 24‐month‐old rats was significantly attenuated (∼15%). In 2‐month‐old rats, when tubulin, polymerized with the hydrolysis‐resistant photoaffinity GTP analogue [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Giα. Transfer of AAGTP from tubulin to Giα was reduced in 24‐month‐old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Giα in cortex membranes was significantly decreased in 24‐month‐old rats. No differences were observed in the amounts of Gsα, Giα, or Gβ subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2‐month‐old and 24‐month‐old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24‐month‐old rats.