In dietary phosphate (Pi) deprivation and in aging there is an inverse correlation between renal proximal tubular brush border membrane (BBM) cholesterol (Chol) content, BBM fluidity, and BBM sodium gradient-dependent Pi transport activity (Na-Pi cotransport). The purpose of this study was to determine whether in vitro enrichment of renal BBM with Chol has a direct modulating effect on Na-Pi cotransport. 12 and 24 mol % increases in Chol content caused dose-dependent decreases in Na-Pi cotransport activity, 2,000 in control, vs. 1,450 in Chol (+12%), vs. 900 pmol/5 s/mg BBM protein in Chol (+24%), all P less than 0.01, which was paralleled by dose-dependent increases in the fluorescence anisotropy of diphenylhexatriene, rDPH, i.e., decrease in BBM fluidity, 0.203 in control, vs. 0.210 in Chol (+12%), vs. 0.219 in Chol (+24%), all P less than 0.01. We found that increasing ambient temperature, which increases BBM fluidity independent of changes in Chol content, increased Na-Pi cotransport. When Na-Pi cotransport was analyzed as a function of BBM fluidity, 1/rDPH, we found that at an equivalent BBM fluidity BBM Chol enrichment still resulted in a dose-dependent decrease in Na-Pi cotransport. Finally, in BBM isolated from rats fed a low Pi diet in vitro enrichment with Chol completely reversed the adaptive increases in Na-Pi cotransport and fluidity. Our study therefore, indicates that Chol is a direct modulator of renal BBM Na-Pi cotransport activity, and that in vivo alterations in BBM Chol content most likely plays an important role in the regulation of renal tubular Pi transport.