The aim of the present study was to investigate if and how microfilaments and microtubules could be involved in the early events of ACTH action. In primary cultures of rat glomerulosa cells, a 30-min preincubation with either 10 microM colchicine (a microtubule-disrupting agent) or 10 microM cytochalasin B (a microfilament-disrupting agent) decreased ACTH-induced cAMP production. Moreover, colchicine decreased cAMP production induced by fluoroaluminate (a nonspecific activator of all G proteins), but not of forskolin (which directly activates adenylyl cyclase). These results indicate that microtubules appear to be essential for the GS protein activation. In contrast, cytochalasin B decreased the stimulating effect of both fluoroaluminate and forskolin, indicating that microfilaments may be involved in both GS and adenylyl cyclase activations. Analyses of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin indicated that the alpha S-subunit of the GS protein was associated with both structures. Stimulation of cells with ACTH induced a rapid increase (within 1 min) in the levels of microfilaments, microtubules, and alpha S associated with the membrane. In addition, ACTH stimulation of cAMP production was very sensitive to Ca2+, without any stimulation in Ca(2+)-free medium. Under these conditions, actin filaments were short and formed a dense network. These observations suggest that the Ca(2+)-free medium stabilized the actin fibers in such a way that activation by ACTH failed, further documenting the importance of microfilaments in cAMP production.