The rate of proteolytic degradation of rabbit skeletal muscle actin by trypsin, alpha-chymotrypsin, and, mainly, subtilisin was followed by dual wavelength spectroscopy at 285 nm by reference at 320 nm. Phalloidin and phallacidin, two toxic bicyclic heptapeptides from the mushroom Amanita phalloides, protect F-actin against degradation by the proteolytic enzymes. G-actin, which does not combine with phalloidin when maintained in the monomeric state by working at low ionic strength, and bovine serum albumin, which also has no affinity to the toxin, are hydrolyzed at the same rates in the presence or absence of phalloidin. The proteolysis of F-actin is distinctly retarded by KCl alone, i.e., without phalloidin, whereas Mg2+ or Ca2+ as sole cations permit a rather high rate of hydrolysis. An even faster degradation of F-actin by subtilisin is observed in the presence of Mg2+ plus cytochalasin B. Adenosine diphosphate and triphosphate have no influence on the rate of the enzymatic degradation. The S sulfoxide of phalloidin, the nontoxic diastereomer of the toxic R form, exerts only a limited inhibitory effect on the enzymatic hydrolysis; secophalloidin, another nontoxic derivative, which does not bind to F-actin has practically no effect.