This report describes the influence of fluid flow and osmotically induced volume changes on Na+–Ca2+ exchange (NCX) activity in transfected CHO cells. Exchange activity was measured as Na+‐dependent Ca2+ or Ba2+ fluxes using the fluorescent probe fura‐2. When exchange activity was initiated by superfusing Ba2+‐containing solutions over the cells for a 20 s interval, a high rate of Ba2+ uptake was observed while the solution was being applied but the rate of Ba2+ uptake declined > 10‐fold when the solution flow ceased. Ba2+ efflux in exchange for extracellular Na+ or Ca2+ (Ba2+–Ca2+ exchange) was similarly biphasic. During NCX‐mediated Ca2+ uptake, a rapid increase in cytosolic [Ca2+] to a peak value occurred, followed by a decline in [Ca2+]i to a lower steady‐state value after solution flow ceased. When NCX activity was initiated by an alternate procedure that minimized the duration of solution flow, the rapid phase of Ba2+ influx was greatly reduced in magnitude and Ca2+ uptake became nearly monophasic. Solution superfusion did not produce any obvious changes in cell shape or volume. NCX‐mediated Ba2+ and Ca2+ influx were also sensitive to osmotically induced changes in cell volume. NCX activity was stimulated in hypotonic media and inhibited in hypertonic media; the osmotically induced changes in activity occurred within seconds and were rapidly reversible. We conclude that NCX activity is modulated by both solution flow and osmotically induced volume changes.