We previously showed an inverse correlation between membrane cholesterol content and Na/Pi cotransport activity during the aging process and adaptation to alterations in dietary Pi in the rat. The purpose of the present study was to determine if alterations in cholesterol content per se modulate Na/Pi cotransport activity and apical membrane Na/Pi protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with -methyl cyclodextrin (- MCD) resulted in a significant increase in Na/Pi cotransport activity accompanied by a moderate increase in apical membrane Na/Pi protein abundance and no alteration of total cellular Na/Pi protein abundance. Conversely, acute cholesterol enrichment achieved with -MCD/cholesterol resulted in a significant decrease in Na/Pi cotransport activity with a moderate decrease in apical membrane Na/Pi protein abundance and no change of the total cellular Na/Pi protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na/Pi cotransport activity and apical membrane and total cellular Na/Pi protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by Laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein (LDL), resulted in parallel and significant decreases in Na/Pi cotransport activity and apical membrane and total cellular Na/Pi protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na/Pi cotransport activity by diverse mechanisms that also include significant interactions of Na/Pi protein with lipid microdomains.