Human lactase-phlorizin hydrolase [EC 3.2.1.23-3.2.1.62] is a disaccharidase located in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a precursor protein in the endoplasmic reticulum and in addition to being glycosylated is subsequently proteolytically processed to the mature microvillus membrane-bound form after passing the trans-Golgi compartment. We studied the oligomerization of human lactase-phlorizin hydrolase in transfected polarized Madin Darby canine kidney cells using metabolic labeling and sucrose-density centrifugation analysis. We detected high mannose dimers of the lactase-phlorizin hydrolase precursor molecule after metabolic labeling with [35S]methionine at 37 and 15 degrees C. In addition, both complex-glycosylated lactase-phlorizin hydrolase precursor molecule and the mature microvillus membrane-bound enzyme showed this oligomeric structure. Chemical crosslinking resulted in the detection of covalently crosslinked lactase-phlorizin hydrolase dimers after sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results provide evidence that oligomerization of lactase-phlorizin hydrolase is an early event and begins in the endoplasmic reticulum.