This experiment had two objectives: to develop an analytical technique to measure the density of digesta and fecal samples; to determine whether this measurement is physiologically relevant. To address the latter question, rats were provided with one of five high-fiber diets or a fiber-free control diet, and density determinations were made on samples from the cecum, proximal and distal colon, and from a passed fecal pellet. The density of colonic contents varied with the fiber component of the diet. The fiber-free control diet produced the densest stool at all sites studied (P < 0.01), whereas wheat bran produced the least dense stool at every site (P < 0.01). There were also density differences among bowel positions within diet treatments: the stool from these animals consuming the fiber-free, guar and pectin diets became denser as it moved distally; wheat bran, on the other hand, produced the opposite effect-the more distal the stool traveled, the less dense it became. Since the density of colonic contents varied with respect to both diet and anatomical site, these data suggest that the traditional technique of expressing the concentration of a constitutentof interest (e.g., bile acid, volatile fatty acid) per gram of digesta or fecal matter may not always be appropriate. This is particularly true if exposure of the intestinal mucosal cells to a substance in the digesta or fecal stream is of interest, since density, rather than msss, reflects the diluting potential of the diet.