The cytotoxic effect of caffeine on myocardial cells was evaluated in primary cultures of rat heart muscle (M) and endothelioid (E) cells. The cells were exposed to 5, 10, or 20 mM caffeine for 6, 12, or 24 h. As an index of cell injury produced by the treatments, in situ changes in lysosomal membrane permeability were measured by a sensitive cytochemical technique. The highest concentration of caffeine (20 mM) produced such toxic signs as reduced cell viability, increased vacuolization, and pseudopod formation. At the lower concentrations, caffeine labilized lysosomal membranes of M cells more easily than those of E cells. Pretreatment with hydrocortisone prior to exposure to caffeine protected E and M cell lysosomes from the labilizing effects of caffeine.