Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca²⁺ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 µmol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP^-/-). During voltage clamp ramps with [Ca²⁺]_i buffering, block of reverse mode block was slightly larger than of forward mode (by 25±5%, P<0.05). In the absence of [Ca²⁺]_i buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca²⁺ influx and Ca²⁺ efflux were reduced to the same extent (to 36±6% and 32±4%, respectively). With normal SR function the reduction of inward NCX current (I_NCX) was 57±10% (n=10); during large caffeine-induced Ca²⁺ transients, it was larger (82±3%). [Ca²⁺]_i transients evoked during depolarizing steps increased (from 424±27 to 994±127 nmol/L at +10mV, P<0.05), despite a reduction of I_CaL by 27%. Resting [Ca²⁺]_i increased; there was a small decrease in the rate of decline of [Ca²⁺]_i. SR Ca²⁺ content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP^-/- mice, in which SR Ca²⁺ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca²⁺. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of I_NCX reduction against effects on Ca²⁺ handling by refining mode dependence and/or including additional targets.