We have studied the contribution of the C-terminus of actin to filament stability by chemical modification and limited proteolysis. Formation of mixed disulfides of the penultimate C-terminal cysteine residue 374 with various low-molecular-mass thiols resulted in filament destabilization, as reflected by an increase in critical concentration and steady-state ATPase activity. These effects were fully reversed by the addition of phalloidin. Both the destabilization by glutathionylation and the reversal of it by phalloidin exhibited a high degree of cooperativity; half-maximal destabilization required the modification of four out of five actin subunits, and half-maximal restabilization by phalloidin was already reached when only one out of 20 actin subunits was complexed. C-terminal truncation by limited trypsinolysis of filamentous actin resulted in a similar destabilization of the polymer, as shown by a 2-3-fold increase in the steady-state ATPase activity. This effect was likewise cooperative and could be reversed by phalloidin. Since truncation of the C-terminus of actin has an effect on stability similar to that of chemical modification with bulky substituents, the possibility can be excluded that, in the latter case, destabilization was caused by steric hindrance. Rather, it seems that the highly conserved C-terminal part of actin plays an active role in establishing a tight contact between neighbouring subunits.