IDDM patients with incipient and overt nephropathy have been found to exhibit an overactivity of RBC sodium-lithium countertransport. To explore the physiological relevance of this finding, we measured the activity of Na⁺/H⁺ antiport in serially passaged cultured skin fibroblasts from IDDM patients with and without nephropathy and from normal, nondiabetic control subjects. Na⁺/H⁺ antiport activity (measured as the rate of amiloride-sensitive Na⁺ influx at pH₁ = 6.4, extracellular pH = 8.0, and [Na⁺] = 1 mM) was elevated significantly in IDDM patients with nephropathy compared with IDDM patients without nephropathy and nondiabetic control subjects (13.35 ± 3.8 vs. 8.54 ± 2.0 vs. 7.33 ± 2.3 nmol Na⁺ · mg protein⁻¹ · min⁻¹; P < 0.006 and P < 0.001, respectively). A kinetic analysis of Na⁺/H⁺ antiport activity showed that the raised activity in IDDM patients with nephropathy was caused by an increased V_max for extracellular Na⁺. K^m values were similar in the three groups. pH-stimulated amiloride-sensitive Na⁺ influx also was higher under baseline conditions and after serum stimulation in cells from IDDM patients with nephropathy. pH^I values were significantly higher, both during active proliferation and after 10-min exposure to serum, in cells from IDDM patients with nephropathy, compared with IDDM patients without nephropathy and nondiabetic control subjects. Serum-stimulated incorporation of [³H]thymidine into DNA was greater in IDDM patients with nephropathy than in the other two groups (35.7 ± 18.9- vs. 17.4 ± 7.5- vs. 11.9 ± 8.7-fold stimulation above baseline; P < 0.01 for both). These data from cultured skin fibroblasts suggest that intrinsic abnormalities in cell function, independent of the metabolic disturbances of diabetes, are characteristic of IDDM patients who develop nephropathy.