This study was undertaken to investigate the mechanisms of beta-lactoglobulin transport and its regulation in rabbit intestinal mucosa. beta-Lactoglobulin, the major bovine milk whey protein, was slightly degraded by ileal brush-border membranes, indicating the presence of phosphoramidon-insensitive neutral endoproteases. Modulation of 14C-radiolabeled beta-lactoglobulin transport across rabbit ileum mounted in Grass diffusion cells was studied in Ringer solution in the presence and absence of (in mM) 25 glucose, 10 galactose, and 0.5 colchicine. The transport was also measured in Dulbecco's modified Eagle's medium as a more "balanced" medium. The transport of the degraded products and of [3H]polyethylene glycol-4000, which was used as an extracellular marker, was enhanced by addition of glucose, presumably indicating passage through the tight junctions. The transepithelial passage of intact protein was estimated to be 10-20% by trichloroacetic acid precipitation and high-performance liquid chromatography, thus indicating the effective transport of the intact protein. The passage of the intact protein was inhibited by both colchicine, a microtubule-depolymerizing agent, and galactose by 50 and 30%, respectively. Galactose-like glycoconjugates, known to be present on the membrane, might be involved in the interaction between the protein and the brush border.