Changes in membrane fluidity have been shown to alter the sodium- dependent renal transport of glucose and phosphate; however, this has not been examined for sodium/sulfate cotransport in the renal proximal tubule. Sodium/sulfate cotransport regulates the homeostasis of sulfate in mammals. The objective of this study was to investigate the influence of alterations of membrane fluidity on sodium-coupled sulfate transport in the Madin-Darby canine kidney cells, which have been stably transfected with sodium/sulfate cotransporter (NaSi-1) cDNA (MDCK-Si). Preincubation of cells with 0.2 mM cholesterol significantly decreased the V(max) for sodium/sulfate cotransport (13.69 ± 1.11 vs 10.15 ± 1.17 nmol/mg protein/5 min, mean ± SD, n = 4, p< 0.01) with no significant alteration in K(m). The addition of benzyl alcohol (20 mM) to cells increased the V(max) of sulfate uptake by 20% (11.97 ± 0.91 vs 14.35 ± 0.56 nmol/mg protein/5 min, mean ±SD, n = 3, p < 0.05) with no significant change in K(m). Membrane fluidity, as measured by the fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH), was significantly increased in MDCK-Si cells treated with 20 mM benzyl alcohol and decreased in the cells preincubated with 0.2 mM cholesterol, compared with control cells. Our results suggest that alterations in membrane fluidity that may occur as a result of disease states, aging, and pregnancy may play an important role in the modulation of renal sodium/sulfate cotransport.