To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs. Examination of total fatty acids in both membranes using gas-liquid chromatography analysis of fatty acid methyl esters showed no difference in the mole percents of the major fatty acids. Our data suggest that changes in bulk membrane fluidity of the Fanconi syndrome dog renal brush border as measured by 1,6-diphenyl-1,3,5-hexatriene cannot be attributed to phospholipid and fatty acid compositional change. In the membranes isolated from affected dog kidney, the cholesterol content determined by gas-liquid chromatography analysis was 66 mol% higher than in membranes isolated from normal dog kidney. This correlates with the higher cholesterol to phospholipid molar ratio of 0.82 +/- 0.08 in the affected animal as compared with 0.58 +/- 0.04 in the normal. Cholesterol content and its microdomain in the membrane bilayer may be important in modulating transport functions. Increased membrane cholesterol content may affect the conformational motility of membrane transport proteins and thus affect their function.