In suspended mast cells, histamine release induced by antigen, PMA and compound 48 was inhibited byC. botulinum C2 toxin, which depolymerizes actin by ADP-ribosylation. In contrast, A23187-induced secretion was not affected by toxin treatment. C2-induced inhibition was paralleled by depolymerization of F-actin. Attachment of mast cells to plastic or glass reduced secretion and increased submembranous F-actin. Under these conditions, C2 toxin increased mediator-induced secretion. C.difficile toxin B, which inactivates F-actin-regulating small GTP-binding proteins of the Rho family, inhibited antigen-compound48 -, PMA- and A23187-induced histamine release in suspended and adherent mast cells. The data indicate diverse functions of the actin cytoskeleton and a crucial role of Rho protein in stimulus-secretion-coupling in mast cells.