The human proximal tubule (HPT) is the characteristic site within the kidney that mediates absorption of glucose. This study was designed to determine whether cultured HPT cells would respond to a hyperglycemic environment through activation of the polyol pathway. The results of this study clearly indicate that exposure of the HPT cells to an extracellular glucose concentration greater than or equal to 11 mM results in substantial intracellular accumulation of sorbitol. This accumulation is inhibited by approximately 70% by treatment with 100 microM sorbinil. When cells growing 24 h on 27.5 mM glucose were changed to medium containing 5.5 mM glucose, sorbitol concentration returned to the control level within 12 h. The activity of aldose reductase was increased by a factor of 1.6 by exposure to elevated glucose concentrations, and the relative reactivity of the enzyme with glucose as substrate was approximately 0.1 compared with that of glyceraldehyde as substrate. Together, these results indicate that cultured cells derived from the HPT undergo activation of the polyol pathway when exposed to a hyperglycemic environment.