Prior studies have established that the expression of the human apolipoprotein B (apoB) gene in the intestine is dependent on DNA sequences located a great distance from the structural gene. To identify the location of those sequences, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate the 5'- or 3'-flanking sequences from a 145-kilobase (kb) bacterial artificial chromosome spanning the entire human apoB gene. Seven RARE cleavage- modified bacterial artificial chromosomes with different lengths of flanking sequences were used to generate transgenic mice. An analysis of those mice revealed that as little as 1.5 kb of 3' sequences or 5 kb of 5' sequences were sufficient to confer apoB expression in the liver. In contrast, apoB gene expression in the intestine required DNA sequences 54-62 kb 5' to the structural gene. Those sequences retained their ability to direct apoB expression in the intestine when they were moved closer to the gene. These studies demonstrate that the intestinal expression of the apoB gene is dependent on DNA sequences located an extraordinary distance from the structural gene and that the RARE cleavage/transgenic expression strategy is a powerful approach for analyzing distant gene-regulatory sequences.