Tunicamycin is a nucleoside antibiotic that inhibits protein glycosylation and palmitoylation. The therapeutic use of tunicamycin is limited in animals because of its toxic effects, particularly in cerebral vasculature. Tunicamycin decreases palmitoylation of the endothelial isoform of nitric oxide synthase, stimulates nitric oxide synthesis, and increases the concentration of intracellular calcium ([Ca²⁺]_i) in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton. FASEB J. 11: A110, 1997). In the present study, we investigated the mechanism by which tunicamycin alters [Ca²⁺]_i using the Ca²⁺-sensitive dye fura 2. We found that tunicamycin increased [Ca²⁺]_i without increasing levels of inositol phosphates. When cells were incubated in the absence of extracellular Ca²⁺, [Ca²⁺]_i rapidly rose in response to tunicamycin, although a full response was not achieved. The pool of intracellular Ca²⁺ mobilized by tunicamycin overlapped with that mobilized by thapsigargin. Extracellular nickel blocked a full response to tunicamycin when cells were incubated in the presence of extracellular Ca²⁺. The effects of tunicamycin on [Ca²⁺]_i were partially reversed by washing out the drug, and the remainder of the response was inhibited by removing extracellular Ca²⁺. These results indicate that tunicamycin mobilizes Ca²⁺ from intracellular stores in a manner independent of phospholipase C activation and increases the influx of Ca²⁺ across the plasma membrane.