A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent-insoluble complexes commonly known as glycolipid "rafts". Thus, aminopeptidase N (EC 188.8.131.52), aminopeptidase A (EC 184.108.40.206), dipeptidyl peptidase IV (EC 220.127.116.11), and sucrase-isomaltase (EC 18.104.22.168-10) were 34-48% detergent-insoluble. Maltase-glucoamylase (EC 22.214.171.124) was markedly less detergent-insoluble (20%), and lactase-phlorizin hydrolase (EC 126.96.36.199-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form, and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment was apparent after only 1 h of labeling, where aminopeptidase N, sucrase-isomaltase, and alkaline phosphatase together comprised 25-30% of the total labeled, detergent-insoluble proteins, showing that sorting of newly made brush border membrane proteins into the glycolipid "rafts" does take place intracellularly. I therefore propose that, in the enterocyte, the brush border enzymes are targeted directly from the trans-Golgi network toward the apical cell surface.