The Ca2+/calmodulin-dependent phosphorylation of neuronal cytoskeletal proteins was studied in brain supernatants prepared from rats exposed via inhalation to 600 to 800 ppm carbon disulfide (CS2) for 14 days. Exposure to CS2 resulted in increased phosphorylation of endogenous MAP-2 and exogenously added neurofilament triplet proteins. There also was an observed increase in the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Slight increases in the binding of a monoclonal antibody to the alpha subunit of CaM kinase II were seen, while large increases in the binding of [125I]calmodulin to the alpha subunit of CaM kinase II also were observed. The finding of large increases in the autophosphorylation and calmodulin-binding to CaM kinase II with only slight increases in the amount of antibody-binding suggests that CS2 exposure results in increased Ca2+/calmodulin-dependent phosphorylation of proteins by inducing an increase in kinase activity.