Phospholipids accumulate within the lysosomes of various cells from individuals taking amiodarone. Studies on cultured cells suggest that inhibition of lysosomal phospholipase A1 and phospholipase A2 by amiodarone may be responsible for this derangement in phospholipid metabolism. Inhibition of lysosomal phospholipases by amiodarone has been suggested as a mechanism of its toxicity, but this relationship has not been clearly established. To examine this question, membrane phospholipids of cultured bovine pulmonary artery endothelial cells (BPAEC) were labeled with 14C-stearic acid, 3H-arachidonic acid, 14C-choline, or 14C-ethanolamine. Radiolabeled BPAEC were then exposed to various concentrations of amiodarone, and endothelial phospholipase activity was measured by isolating and quantifying various phospholipase products. These findings were compared to a standard indicator of endothelial cytotoxicity using 51Cr release. Six-hour exposures to 5 to 20 micrograms/ml amiodarone produced no BPAEC toxicity and were accompanied by some evidence of decreased phospholipid hydrolysis. At concentrations above 20 micrograms/ml, amiodarone caused significant BPAEC toxicity as indicated by 51Cr release, and this was closely associated with the liberation of substantial amounts of 3H-arachidonic acid and 14C-stearic acid from phosphatidylcholine and phosphatidylethanolamine. In BPAEC labeled with 14C-choline and 14C-ethanolamine, cytotoxic doses of amiodarone caused accumulations of 14C-phosphocholine and phosphorylethanolamine, expected products of phospholipase C, but without increases in phospholipase A products. We conclude that exposure of BPAEC to toxic concentrations of amiodarone is associated with extensive hydrolysis of phosphatidylcholine and lysophosphatidylethanolamine via a phospholipase C-specific mechanism, and suggest that this may be a mechanism in the pathogenesis of amiodarone toxicity.