Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca 2 + ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC 5 0 s between 5 and 50μM. It inhibited more potently the purified Ca 2 + ATPase activity from skeletal muscle with an IC 5 0 of 5μM. Detailed effects of this inhibitor on the Ca 2 + and ATP dependence upon activity indicate that it affects the high-affinity Ca 2 + -binding (E1) form of the ATPase. In addition, paxilline is a ''competitive'' inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site K m , without affecting the catalytic binding site K m . At higher concentrations, paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that paxilline has two effects on the Ca 2 + ATPase. At lower concentrations (5-10μM), paxilline inhibits the ATP-dependent acceleration of Ca 2 + release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, paxilline inhibits phosphoenzyme formation.