The transepithelial absorption of food-type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo-2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical-basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4°C (7.41 ± 1.26 pmoles/h·cm2 vs. 27.40 ± 8.91 at 37°C). The amount conveyed via the direct pathway was very small (0.89 ± 0.35 pmoles/h·cm2) and did not diminish at 4°C (1.43 ± 0.59 pmoles/h·cm2). In the basal-apical direction, HRP transport along the degradative pathway at 37°C was similar to the apical-basal value and was inhibited at 4°C (16.40 ± 4.50 vs. 2.72 ± 2.52 pmoles/h·cm2), but along the direct pathway, it was eight times the apical-basal value (8.36 ± 3.11 pmoles/h·cm2) and was inhibited at 4°C (2.43 ± 0.78 pmoles/h·cm2). Intact HRP fluxes were not correlated with the eletrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10−5 M did not affect direct or degradative transport in the apical-to-basal direction. These results suggest that in CaCo-2 cells HRP undergoes bidirectional transcytosis by a fluid-phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.