Abnormal erythrocyte sodium-lithium countertransport (Na-Li CT) activity, traditionally measured at a single sodium concentration of 140 mmol.l-1 (V140), may represent an inherited risk marker for diabetic nephropathy. The membrane defect underlying this association is poorly understood, though modulation by key protein thiol groups appears to be important in essential hypertension. To improve understanding of this abnormality, Na-Li CT kinetics in untreated erythrocytes and after thiol group alkylation with N-ethylmaleimide were investigated in 18 subjects with diabetic nephropathy, 20 normoalbuminuric insulin-dependent diabetic (IDDM) subjects and 18 non-diabetic individuals. Using the traditional assay, V140 was similar in subjects with diabetic nephropathy compared to IDDM control subjects vs 0.311 (0.152-0.475) (0.247 (0.111-0.498) mmol Li.h-1.l erythrocytes-1). Kinetic parameters were abnormal in subjects with diabetic nephropathy compared with diabetic and non-diabetic control subjects with both Vmax (maximal Na-Li CT activity) (0.454 (0.257-0.963) vs 0.338 (0.183-0.972) vs 0.332 (0.213-0.603) mmol Li.h-1.l erythrocytes-1, p < 0.05), and Vmax/Km(So) ratio, reflecting ion association (6.03 (2.3-9.6) vs 4.73 (2.0-10.4) vs 4.48 (1.5-7.1), p < 0.01), significantly higher. N-ethylmaleimide decreased K(m)(So) and Vmax abolishing differences in Vmax/Km(So) ratio between groups (2.45 (1.18-4.21) vs 2.23 (0.96-4.3) vs 2.44 (1.4-3.7), but enhancing the differences in Vmax (0.186 (0.090-0.315) vs 0.120 (0.051-0.256) vs 0.128 (0.080-0.206) mmol Li.h-1.l erythrocytes-1, p < 0.0001). Of subjects with diabetic nephropathy, 78% were outside the 75th percentile of the non-diabetic control subjects when Vmax and Vmax/Km(So) ratio were combined, compared to 20% of the normoalbuminuric control subjects. We conclude that the traditional assay, V140, is poor at detecting individuals with diabetic nephropathy. Study of the kinetic parameters of the transporter, including thiol group modulation, suggests that increased ion association, Vmax/Km(So) ratio may represent the inherited defect and improves identification of subjects with diabetic nephropathy.