Hypotonic treatment of cultured rat hepatocytes significantly decreased the monomeric G-actin level by 18% after 120 min while the level of filamentous F-actin remained essentially unchanged. Simultaneously the level of cellular actin mRNA was increased by 53%. Incubation of hepatocytes for 120 min with the F-actin stabilizing toxin phalloidin from Amanita phalloides led to a decrease of G-actin by 70% and an increase of F-actin by 55%. Although the toxin dependent decrease of G-actin was much more pronounced than the decrease after hypotonic treatment, the increase of actin mRNA was similar under both conditions. Simultaneous treatment with hypotonic medium did not result in a further decrease of the G-actin level. On the other hand, the G-actin elevating C2 toxin from Clostridium botulinum completely blocked the effects of osmotic stress on G-actin and actin-mRNA content. The results demonstrate that already an essentially physiological decrease of G-actin without alterations of F-actin results in a substantial enhancement of the actin mRNA level, indicating the physiological significance of this autoregulation.