Natural organic matter (NOM) is an important constituent of aquatic environments; however, its influence on aquatic biota remains poorly studied. In the current study, NOM was isolated from nine different sites in southern Ontario, Canada, by the on-site treatment of water by reverse osmosis, followed by cation exchange. NOM from each site was reconstituted to 10 mg of C/L and pH 7.0 and exposed to either adult rainbow trout implanted with indwelling catheters or to in vitro primary cultures of the gill epithelium grown on semipermeable membranes. In both the in vivo and in vitro preparations, NOM was found to hyperpolarize transepithelial potential (TEP), with the magnitude of this change correlating extremely well to the absorptivity of the NOM at 340 nm, which is an index of its aromaticity. Gill hyperpolarization appeared to be independent of Ca2+ complexation by the NOM in all but two samples tested. We argue that NOM has direct actions on the ionic transport and/or permeability properties of fish gills. While NOM effects on the bioavailability of contaminants are well-known, NOM actions on such fundamental physiological properties of the gills have previously been overlooked. These may be of comparable or greater magnitude than commonly reported for other water-quality variables (e.g., hardness, pH, salinity) and therefore of critical importance in ecological understanding and risk assessment.