The mechanism of increase in intracellular Ca2+ concentration ([Ca2+]i) by removal of extracellular Na+, which phenomena were reported previously (Japan. J. Pharmacol. 63 83-91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca2+]i. The pretreatment of thapsigargin (0.5 microM), but not nicardipine (10 microM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca2+ (nominally Ca2+-free solution), the Na+-free induced transient increase was observed, but neither the second cell exposure to the Na+-free solution nor the following application of histamine increased [Ca2+]i, indicating that removal of extracellular Na+ releases Ca2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP3)-releasable pools. The Na+-free induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca2+ and releasable Ca2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca2+ by the reverse-mode action of the Na+/Ca2+ exchanger can release Ca2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools.