C. difficile toxin B is a potent cytotoxin known to disrupt the microfilaments of cultured cells. We have recently shown also increased phospholipase A2 activity in cells treated with toxin B. The activity was detected as a toxin-induced, dose-dependent release of 14C-arachidonic acid from prelabeled fibroblasts. Here is shown that the toxin elicited a 14C-arachidonic acid release in a cell mutant resistant to the toxin B effect on the microfilaments. The toxin-induced release was further characterized using fibroblasts. Within 20 min high doses of toxin B (6 micrograms/ml) elicited a release which increased exponentially with time. Of the major membrane phospholipids the lipase activity affected mainly phosphatidyl ethanolamine. Neither cycloheximide nor pertussis toxin treatment or target cells inhibited the toxin-induced release, while it could be increased with 12-O-tetradecanoylphorbol-13-acetate. Our results also suggest a toxin-mediated increase in phospholipase C activity occurring at a later stage than the phospholipase A2 activation. We conclude that the ability of toxin B to induce phospholipase activation represents a hitherto unrecognized toxin B effect which is neither a cause nor a consequence of toxin-induced microfilament disorganization.