Interferon-γ (IFN-γ) at a concentration of 50 U/ml increased internal Ca 2+ in the monocyte-like cell line U937 by about 100% within 3 min of addition, as determined by indo-1 fluorescence. This IFN-γ-induced increase was reduced to 30–40% of basal (Ca 2+) i by the addition of diltiazem (1 uM) or incubation in Ca 2+-free buffer. A crude membrane preparation obtained by differential centrifugation of sonicated U937 cells possessed Ca 2+-ATPase activity (10 nmol ATP hydrolyzed/min/mg protein at 30 C) and sequestered Ca 2+ to a level of 8 nmol/mg protein in 30 min. Addition of inositol trisphophate (IP 3) (10 uM) after accumulation of Ca 2+ resulted in release of a portion of the sequestered Ca 2+ within 30 s, which was then resequestered. Although mitochondrial contamination was indicated by partial inhibition of Ca 2+ uptake by oligomycin A, this mitochondrial inhibitor had no effect on the IP 3-induced Ca 2+ release. These results suggest that the increase in U937 cell cytoplasmic Ca 2+ induced by IFN-γ results from both intacellular redistribution of Ca 2+, probably via polyphosphoinositide metabolism, and the entry of extracellular Ca 2+ through slow channels.