General anesthetics, either alone or in combination, can be detrimental to the developing mammalian brain and induce extensive apoptotic degeneration of immature neurons when they are administered at the peak of synaptogenesis. Because neuron development and normal functions depend on the integrity of astroglia, we sought to determine whether general anesthesia also causes disturbances in the early development of astroglia. Using isoflurane, an inhaled anesthetic that is highly toxic to immature neurons, we studied primary astroglia cultures, focusing on very early development (Day-In-Vitro 4 treatment). Exposure to 3% isoflurane for 24 hours delayed morphological differentiation and impaired the growth of immature astrocytes. The timing of delayed astroglia maturation and growth coincided with a major disturbance in actin cytoskeleton sculpting that was manifest as impaired actin stress fiber formation and cytoskeletal organization and downregulation of the focal adhesion protein, paxillin. Isoflurane-induced actin cytoskeletal changes were accompanied by a significant decrease in protein levels of the endogenous GTPase RhoA that regulates the phosphorylation of myosin light chain protein, suggesting that isoflurane-induced impairment in glial growth and morphological development is, in part, mediated by the RhoA/myosin light chain protein signaling pathway.