Na+/H+ exchanger 1 (NHE1) is a ubiquitous membrane transporter that regulates pHi, [Na+]i and cell volume. In the heart, stretch-activation of NHE1 has been claimed to be a key element of the slow force response to stretch1. By raising pHi and [Na+]i, NHE1 activation may initiate hypertrophy2. We examined osmotic activation of NHE1 and the related Na+/HCO3- cotransporter (NBC) in rat isolated ventricular myocytes. Adult rat ventricular myocytes were enzymatically isolated. pHi was imaged using cSNARF-1. 160 mM sucrose was added to increase osmolarity. Data are mean + SEM, compared with unpaired Student’s t-test. Intrinsic buffering capacity (βint) was measured by ammonium removal. Hyperosmotic shrinkage increased βint 1.6-fold (26.3+1.6mM (n=17) vs. 16.1+1.9mM (n=6) at pH 7.3, P<0.01). pHi was increased using an ammonium prepulse and the slope of pHi recovery was used to calculate acid-efflux (J=dpH/dt* β). NHE-mediated acid-efflux was greatly increased in hyperosmotic medium (11.0+0.5mM/min (n=25) vs. 2.7+0.3mM/min (n=8) at pH 7.0, P<0.001), producing an alkaline shift of 0.25 pH units in the pHi-flux relationship. Upon addition of 160 mM sucrose, myocytes showed gradual NHE-mediated alkalinization of 0.3 pH units over 8 min (n=5). This was abolished by the NHE-inhibitor DMA (5-(N,N-dimethyl)amiloride, 30µM, n=7). To elucidate the osmotic regulation of NBC, bicarbonate-buffered solutions were used (NHE inhibited with 30µM DMA). NBC-mediated acid-efflux was 1.8-fold increased in hyperosmotic solution (6.3+0.7mM/min (n=17) vs. 3.4+0.3mM/min (n=25) at pH 7.0, P<0.001). Testing a possible role of carbonic anhydrase (CA) in the osmotic activation of NHE1 and NBC, we found that acid-efflux was not altered by addition of ETZ (100µM) in isosmotic or hyperosmotic bicarbonate-buffered media (isosmotic: 10.5+0.9mM/min (n=21) vs. ETZ 14.1+1.6mM/min (n=6) at pH 6.6, P=0.065; hyperosmotic: 25.9+1.7mM/min (n=15) vs. ETZ 26.7+1.8mM/min (n=5) at pH 6.7, P>0.8). This rules out the necessity of CA activity for the combined osmotic effect on NHE and NBC. Given the strong osmotic effect on NHE, we examined its role in myocyte volume regulation. Both video-imaging and z-stack techniques3 showed a rapid shrinkage in hyperosmotic solution within the first minute without subsequent regulatory volume increase (z-stack data as normalized volume: after 1 min, 81.8+0.7%; 5 min, 81.1+1.0%; 10 min, 79.5+1.3%, n=7). The contribution of NHE to cellular volume maintenance during hyperosmotic shock was found to be negligible (30µM DMA added, compared to above data: after 1 min, 86.6+1.2%, P<0.01; 5 min, 83.9+0.7%, P>0.065; 10 min, 81.2+1.0%, P>0.36, n=5). We conclude that strong activation by hyperosmotic stimuli is a common characteristic of the cardiac acid-extruders NHE1 and NBC, but does not directly involve activation of catalytic activity from native carbonic anhydrase.