Recent observations indicate that Sertoli cell aromatase activity decreases when cultures are performed at high density. Increasing cell density modifies cell shape in culture from flat cells with visible anchorage sites and abundant intercellular spaces to cells with higher profiles that form a uniform epithelial sheet with no intercellular spaces. Changes in cell architecture are associated with reorganization of the cytoskeleton components. In this report, we have tested whether disruption of microfilaments and microtubules by cytochalasin B and colchicine, respectively, has any effect on the ability of FSH to stimulate aromatase activity. Cytochalasin B, but not colchicine, significantly enhanced aromatase activity in FSH and dbcAMP stimulated cells. The increase in aromatase activity was accompanied by a striking change in cell morphology. Time course studies suggested that microfilament organization is involved in some metabolic event which occurs sometime between 2 and 4 h after the initial steps of FSH action. The reversibility of the biochemical and morphological changes induced by cytochalasin B was demonstrated. The effect of cytochalasin B was observed in high but not in low-density cultures, suggesting that microfilament organization in high-density cultures constrains FSH stimulation of aromatase activity. The last two observations made suggest the existence of a dynamic interplay between microfilament organization and FSH action in Sertoli cells.