Fluorescence staining characteristics of "large vacuoles," i.e. vacuoles ranging up to almost cell size, were studied in suckling rats and pigs. In the distal epithelium of the small intestine of suckling rat, yellow autofluorescence and accumulation of orally administered FITC-dextran were observed in the supranuclear vacuole. In both species the weakly basic amino dye acridine orange (AO) stained the nuclei at neutral pH bright yellow-green and the transport and digestive vacuoles bright red or orange. It is concluded that trapping and accumulation of the dye (red shift) were due to the acidity of the vacuolar interior. Assessment of the vacuolar pH in rat enterocytes is in agreement with published data on lysosomal pH values. Acidic buffers, lysosomotropic and destructive agents, or illumination with bright light induced irreversible fading of AO-stained vacuoles; the color of the porcine transport vacuoles was the most labile. This fading was used to differentiate vacuoles from other structures, e.g., vacuolar inclusion bodies and goblet cells. In suckling rat, staining characteristics of the gut epithelium changed on Days 19 and 20 of postnatal age. Detection of acidity in the distal (digestive) vacuoles supports the lysosome-like nature of their function. They appear to constitute an auxiliary, intracellular digestive system for the young animal. However, the function of acidity in the non-digestive transport vacuoles of newborn pig is unclear.