The absorption mechanism of 4 H2O-soluble dyes, methylene blue (MB), bromthymol blue (BTB), bromphenol blue (BPB) and phenol red (PR), from the rat small intestine was investigated. Absorptive characteristics of these dyes, highly ionized compounds of very low lipoid solubilities at the physiological pH range of the small intestine, varied widely. The percentage net absorption of MB, BTB, BPB and PR were 46.0 .+-. 2.9, 14.7 .+-. 3.7, 2.4 .+-. 1.1 and 1.2 .+-. 0.5%, respectively. The degree of binding to the intestinal mucosa preparation determined in vitro was correlated to the disappearance from the intestinal lumen and the accumulation in tissue, rather than net absorption. The binding to brush borders, the 1st step of intestinal absorption, was parallel to the tendency of uptake by isolated epithelial cells. Binding to the mucosa, especially to the brush borders (microvilli) as the 1st step, is important in the process of absorption of these H2O-soluble dyes from the rat small intestine. The poor absorbability of PR is due to its very low affinity to the intestinal mucosa in addition to poor lipoid solubility.