The effects of the ionophoric antibiotic X537A on cell structure were studied with phase-contrast, fluorescence, and electron microscopy. X537A induced selective vacuolation of the Golgi apparatus of vascular and intestinal smooth muscle, epithelium, plasma cells, and cultured chick heart and guinea pig vascular smooth muscle cells. The swelling of the Golgi apparatus induced by X537A was reversible in the systems examined for reversibility: vascular smooth muscle and cultured chick heart. Myelin figures were common in the Golgi apparatus vacuolated by X537A. Fluorescence microscopy of cultured cells incubated with X537A showed the characteristic blue X537A fluorescence associated with lipid globules in the cultured cells. Incubation of cultured chick heart cells with X537A reduced the beating rate and, after 24-72 h, abolished the sarcomere pattern. The swelling of the Golgi membranes produced by X537A in cultured vascular smooth muscle was associated with inhibition of D-[6-3H]glucosamine and [35S]sulfate incorporation into glycosaminoglycans.