The molecular mechanism of the action of antidepressants beyond the receptor level has not yet been elucidated. We have investigated the effects of long-term treatment with desipramine on the phosphorylation state of microtubule-associated protein 2 (MAP2) and microtubule assembly in the rat cerebral cortex. Phosphorylation of MAP2 was detected by immunoblotting after immunoprecipitation of MAP2 in the soluble fraction. The degree of phosphorylation of serine residues of MAP2 was significantly increased after chronic administration of desipramine without changes in the total concentration of MAP2. Microtubule assembly in crude brain extracts was monitored in terms of changes in turbidity measured at 350 nm using a spectrophotometer. Chronic but not acute treatment with desipramine inhibited microtubule assembly, assayed in the presence of a phosphatase inhibitor, calyculin A, whereas the inhibition was completely nullified in the absence of calyculin A. Desipramine had no direct effect on microtubule assembly in vitro. These results raise the possibility that the changes in the degree of phosphorylation of MAP2 and microtubule assembly represent intracellular modifications involved in functional changes elicited by long-term treatment with desipramine.