The ablation of renal mass and institution of a high protein diet both lead to renal cortical hypertrophy and increased glomerular filtration rate (GFR). We studied Na+ transport in rat microvillus membrane vesicles isolated from uninephrectomized or sham operated rats fed 6% (low), 24% (standard), or 40% (high) protein diets. The feeding of high protein, as compared with low protein, was associated with a 50% increase in rates of pH-stimulated 22Na+ transport in isolated vesicles from sham and uninephrectomized animals. Values for the standard protein diet were intermediate to values for high and low protein. At each level of dietary protein intake, vesicular Na+ transport was greater in the uninephrectomized than in sham rats. The high protein diet was also associated with increased vesicular 22Na+ flux inhibitable by 1 mM amiloride. Increases in total and amiloride sensitive flux were also noted in the absence of a pH gradient. Conductive Na+ and H+ transport were not altered, nor were sodium-glucose and sodium-alanine cotransport. Kinetic studies revealed evidence for an increased Vmax of Na+-H+ exchange in uninephrectomized animals fed a 40 vs. a 6% protein diet whereas Km was unchanged. Supplements of NaHCO3 in the 40% protein diet, to adjust for an increased rate of net acid excretion, did not prevent the increased rates of Na+-H+ exchange. However, treatment with actinomycin D (0.12 mg/kg) prevented the increased Na+-H+ activity as well as the increased renal mass and GFR noted 24 h after unilateral nephrectomy. Na+-H+ exchange rate was closely correlated with GFR (r = 0.961; P less than 0.005) and renal mass (r = .986; P less than 0.001). These observations provide evidence for modification of the luminal membrane Na+-H+ exchanger in response to changes in dietary protein content and nephron number.