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A novel, computer-assisted program was developed to analyze the time course of Na+-glucose cotransport by rat renal cortical brush-border membrane vesicles (BBMV). Transporter characteristics can be measured, which routine kinetic analyses fail to distinguish: cotransporter membrane density is derived from the picomoles of D-glucose bound per milligram of protein. Binding is stereospecific, blocked by phlorizin, and supported equally well by Na+ or K+ (but not Cs+). Quasi-first-order influx and efflux rate constants for the composite Na+-driven influx and the (presumed) Na+-independent efflux processes were highly dependent on glucose concentration. Either two Na+-glucose transporters exist in proximal tubules or a single mechanism abruptly changes rate when glucose falls to low levels. The major operation mode is slow, has a high capacity but low affinity, and may have a 2 Na+:2 glucose stoichiometry (Hill coefficient is unity). The minor system is a fast, smaller-capacity, higher-affinity operation with a 2 Na+:1 glucose stoichiometry that was not distinguishable when the same data were analyzed in conventional kinetic plots. Results with streptozocin-induced diabetic rats illustrate the method's utility. Low-glucose-affinity cotransporters were upregulated in hyperglycemic, but not in cachectic, ketoacidotic animals. Rate constants, especially for efflux, were decreased in diabetes.