Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. Its mechanism of action is not yet well understood. This paper investigates the sites of MC 540-mediated photodamages in L1210 leukemia cells by examining the effects of MC 540-sensitized photoirradiation on several soluble and membrane-bound marker enzymes. When exposed to MC 540 and white light under a standard set of conditions, the activities of Na+/K(+)-ATPase, Mg2(+)-ATPase, and 5'-nucleotidase (three plasma membrane-bound enzymes) were reduced by 54, 49, and 55%, respectively. None of the intracellular enzymes included in this survey was affected by MC 540-sensitized photoirradiation as long as the plasma membrane remained intact. The two soluble enzymes, lactate dehydrogenase and malate dehydrogenase, remained refractory to MC 540-sensitized photoirradiation even after the plasma membrane had been disrupted. By contrast, the activities of the membrane-bound enzymes, NADPH-cytochrome c reductase and succinate dehydrogenase, were reduced in cell lysates by 55 and 81%, respectively. Purified NADPH-cytochrome c reductase was about 3 times less sensitive than the microsomal enzyme, suggesting that the membrane environment facilitated photoinactivation. The MC 540-sensitized photoinactivation of enzymes was accelerated in the presence of deuterium oxide and inhibited if oxygen in the medium was displaced by nitrogen or azide was added to the medium. Taken together, these data support the view that the plasma membrane is a major target of MC 540-mediated photodamages, that the inactivation of membrane-bound enzymes is an oxidative process, and that at least some photodynamic damages are mediated by type II chemistry.