It is generally accepted that cytochalasin B (CB), as well as other cytochalasins, shorten actin filaments by blocking monomer addition at the fast-growing ("barbed") end of these polymers. Despite the predominance of this mechanism, recent evidence suggests that other interactions may also occur between CB and F-actin. To investigate this possibility further we have employed an actin derivative, prepared by substitution at Cys374 by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several ionic conditions. We further show that in the presence of CB the glutathionyl-F-actin exhibits a significantly higher ATPase activity than the non-modified F-actin. These data argue that the incorporation of glutathionyl groups prevents the high-affinity binding of CB to the barbed end of actin filaments, probably due to a decreased hydrophobicity of the CB binding site by the introduction of the hydrophilic glutathionyl residue. Despite the lack of substantial binding at equilibrium, we have found that the addition of CB to glutathionyl-F-actin results in extensive fragmentation of the filaments, as demonstrated by electron microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from barbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cytochalasins and with the physiologically important actin capping/severing proteins.