Insulin was reported to decrease Na+/H+ exchange activity in murine vascular smooth muscle (VSM) tissue. In most other cells, insulin increases activity. We tested the effects of insulin on Na+/H+ exchange activity in primary cultured canine VSM cells. Intracellular pH (pHi) was measured with 2′,7′-bis(2-carboxyethyl)-5-carboxyfluorescein fluorescence and Na+ uptake by isotopic methods. Insulin alone did not significantly affect pHi (7.13 ± 0.05 vs. 7.10 ± 0.03 for control and insulin, respectively; p = not significant), and EIPA alone lowered it to 6.98 ± 0.04 (p < 0.05), upon which insulin lowered it further to 6.91 ± 0.04 (p < 0.05). In the presence of a pHi clamp, pHi/extracellular pH (pHo) 7.1/7.4, insulin increased amiloride-sensitive 22Na uptake by 98 ± 25% (p < 0.05). At pHi/pHo 6.0/7.4 or 6.7/7.4, amiloride-sensitive 22Na uptake was stimulated by 378 ± 59 and 105 ± 27%, respectively, compared to pHi/pHo 7.1/7.4 (p < 0.05 for all 3 versus each other), but was insulin insensitive. In acid-loaded cells (pHi 6.0), addition of extracellular Na+ (pHo 7.4) caused rapid intracellular alkalinization, and the initial rate was not affected by insulin. It is concluded that insulin stimulates acid accumulation in VSM cells that is normally effluxed by increased Na+/H+ exchange activity, and insulin may directly stimulate Na+/H+ exchange activity when cells are clamped at resting pHi but not at acidic pHi.