We recently found that the morphology of dibutyryl cyclic AMP (dbcAMP)-stimulated cultured cerebral hemisphere astrocytes changed from stellate to polygonal within 3 h after methylmercury (MeHg) exposure at 2 and 3 µM. To elucidate the mechanism of this change (spreading) in astrocytes induced by MeHg, the effects of inhibitors of Rho and its downstream effector, Rho kinase, and the expression levels of total and active RhoA were investigated under serum-free conditions in the presence of dbcAMP. Pretreatment with C3 transferase (a Rho inhibitor) completely inhibited the spreading of astrocytes induced by MeHg at both doses for 3 h. However, pretreatment with Y-27632 (a Rho kinase inhibitor) inhibited the spreading induced by MeHg at 2 µM, but not by that at 3 µM. Expression levels of total RhoA were similar under all conditions examined, including in the presence of MeHg. In contrast, the expression level of active RhoA in astrocytes exposed to MeHg at 2 or 3 µM for 30 min was markedly higher than that in astrocytes exposed to solvent alone, although no difference was observed in the level between astrocytes exposed to MeHg at either dose. In addition, the active RhoA levels in MeHg-exposed astrocytes at both doses were similar to the level in astrocytes maintained in 15% serum-containing medium. These results suggest that RhoA activation is involved in the change in shape of astrocytes induced by MeHg at 2-3 µM, and that Rho kinase is, at least partly, related to the shape change in its downstream signal transduction.