Monoacyl and diacyl phospholipids labeled with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) were used to measure the rate of phospholipid transfer between artificial large unilammelar vesicles and rabbit intestine brush-border membrane vesicles (BBMV). This assay demonstrated that transfer into and out of BBMV occurred in two kinetic phases. The fast rate of transfer resulted from interbilayer transfer between the outer membrane leaflets. The slower rate resulted from transmembrane transfer across the BBMV. Treatment of the BBMV with the protease pronase E and with the histidyl reagent diethylpyrocarbonate inhibited the rate of transmembrane transfer. Phospholipids containing one or two acyl chains and a wide range of head-group structures were transported across the membranes with rates that increased as a direct function of their water solubility. These data demonstrated the existence of a protein transporter in the intestinal brush border that functions to absorb slightly water-soluble lysophospholipids and short-chain diacyl phospholipids without redistributing the highly water-insoluble long-chain diacyl phospholipids between the outer and inner leaflets of the brush-border membrane.