The brush border (BB) of the enterocyte is a well-studied example of the actin-based cytoskeleton. We describe here a cell culture model that expresses a faithful representation of the in vivo structure. Two clones (C2BBe 1 and 2) isolated from the cell line Caco-2 (derived from a human colonic adenocarcinoma) formed a polarized monolayer with an apical BB morphologically comparable to that of the human colon. BBs could be isolated by standard methods and contained the microvillar proteins villin, fimbrin, sucrase-isomaltase and BB myosin I, and the terminal web proteins fodrin and myosin II. The immunolocalization of these proteins in confluent, filter-grown monolayers was determined by laser scanning confocal microscopy; patterns of distribution comparable to those in human enterocytes were observed. Sedimentation analysis of cell homogenates derived from C2BBe cells and human colonic epithelial cells demonstrated similar patterns of fractionation of BB proteins; the physical association of those proteins, as determined by extraction from the BB, was also comparable between the two cell types. Like enterocytes of the human intestine, C2BBe cells expressed multiple myosin I immunogens reactive with a head domain-specific monoclonal antibody raised against avian BB myosin I, one of which co-migrated with the approximately 110 kilodalton (kDa) heavy chain of human BB myosin I. In addition, the C2BBe cells express a pair of higher molecular mass immunogens (130 and 140 kDa). These myosin I immunogens all exhibit ATP-dependent association with the C2BBe cytoskeleton. Although the higher molecular mass immunogens were detected in several other human intestinal lines examined, including the parent Caco-2 line, none of these other lines expressed detectable levels of the 110 kDa immunogen, which is presumed to be the heavy chain of human BB myosin I.