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 occured, 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.