The cardiac antidysrrhythmic drug amiodarone can give rise to potentially fatal pulmonary toxicity in large numbers of patients. The effect of amiodarone on Ca 2+ homeostasis and cell injury has been studied using human pulmonary artery endothelia (HPAE) cells in vitro. Amiodarone produced a concentration-dependent increase in intracellular free Ca 2+ concentration ([Ca 2+] i) to micromolar levels that are similar to those seen with physiological stimuli that increase [Ca 2+] i. Unlike physiological stimuli, the rise in [Ca 2+] i produced by amiodarone developed slowly and was maintained over at least 30 min. Omitting Ca 2+ from the external medium reversibly prevented the amiodarone-induced rise in [Ca 2+] i. Amiodarone treatment increased the apparent first order rate constants for 45Ca 2+ influx and efflux in intact HPAE cells. 45Ca 2+ accumulation into the endoplasmic reticulum of saponin-permeabilized HPAE cells was decreased by amiodarone treatment. The release of 45Ca 2+ from the endoplasmic stores by the putative intracellular second messengers inositol-1,4,5-trisphosphate, arachidonic acid, and Ca 2+ was blocked by amiodarone treatment. The changes in Ca 2+ homeostasis coincide with an increase in [ 3H]deoxyglucose release as a measure of early cell injury by amiodarone. It is concluded that amiodarone can produce an increase in [Ca 2+] i by an action on the plasma membrane that allows the influx of external Ca 2+. This increase in [Ca 2+] i, together with other changes in Ca 2+ homeostasis, may be responsible for the early cell injury associated with amiodarone toxicity.