Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 ± 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 ± 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 ± 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 ± 0.04 µmol · h¹ · mg protein¹ at 300 mosmol/kg to 1.81 ± 0.16 at 600 mosmol/kg and to 3.02 ± 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 ± 16 nmol/10 min compared with 389 ± 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.