We investigated the relationship between F-actin damage and cell detachment using nephrotoxic L-cysteine S-conjugates. In vivo S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induced loss of F-actin in the S3 segment of the proximal tubule in the outer stripe of the outer medulla, which was associated with loss of the brush border and loss of cells from the basement membrane. In vitro DCVC caused the detachment of primary cultured rat renal proximal tubular cells (PTC), which was clearly associated with F-actin damage. Disorganization of F-actin correlated with an increase in cellular levels of G-actin, indicating depolymerization of F-actin. Cell detachment was preceded by a complete loss of the alpha-actinin binding protein talin from the focal adhesions, which was directly associated with F-actin disorganization. Inhibition of formation of highly reactive metabolites from L-cysteine S-conjugates by L-cysteine-S-conjugate beta-lyase completely prevented both F-actin damage and cell detachment by DCVC. Although inhibition of DCVC-induced lipid peroxidation and reduction of intracellular free calcium by N,N'-diphenyl-p-phenylenediamine and the acetoxymethyl ester of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, respectively, clearly prevented cell death, no protection was observed against the DCVC-induced F-actin disorganization, talin redistribution, and cell detachment. Also, F-actin damage was unrelated to changes in the energy status of the PTC, since cellular ATP content was unaffected. The data clearly demonstrate a close relationship between F-actin damage, disturbances of focal adhesions, and cell detachment. In addition, different molecular pathways are involved in the cell detachment caused by F-actin disorganization and initiation of cell death.