In the jejunal epithelium of adult animals, horseradish peroxidase (HRP) is transported via two functional pathways: a major route involving lysosomal degradation and a minor route allowing transport of the intact protein. The postnatal development of HRP absorption and the influence of conventional microflora were studied in vitro, using jejunal epithelium of conventional and germ-free suckling mice mounted in Ussing chambers. In conventional mice, tritiated peroxidase ([3H]HRP) transport from mucosa to serosa did not change with age except during days 3 and 4, when it increased fivefold. This increase was entirely due to the rise in intact HRP transport and was not related to a decrease in lysosomal cathepsin B and D activities. In germ-free mice, HRP transport developed similarly but decreased by 75% along both pathways. This reduced absorption was associated with lower cathepsin B and D activities. These results strengthen the existence of two transcellular pathways in nonimmunoglobulin protein absorption by the proximal gut with a limiting step situated before the lysosomal system, presumably at the brush-border membrane. Their development is not parallel and does not correlate with gut closure to immunoglobulin transfer.