Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4°C. Upon phase separation at 32°C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, γ-glutamyl transpeptidase, and Ca 2+Mg 2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.