Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd(2+)-associated cytoskeletal reorganization. Low concentrations of Cd(2+) (0.5-2 μM) caused an increase in actin glutathionylation by 6h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd(2+)-dependent effect, as only Cd(2+) concentrations above 2 μM were sufficient to increase ROS. However, low [Cd(2+)] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd(2+) exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd(2+) concentrations increase ROS levels and prevent an increase in actin-glutathione conjugates.