Chemical-diffusing substrates were designed to allow delivery of toxicants to mature periphyton communities under natural conditions without contaminating the surrounding environment. Artificial stream validation studies were conducted in which the effects of substrate-released copper (Cu) on periphyton communities were compared to those generated in a more conventional manner (via water column additions). Effects of copper on the following community parameters were assessed: total community biomass (measured as ash-free dry mass), relative chlorophyll a (chl a and adenosine triphosphate contents, and relative biomass of heterotrophic bacteria. Exposure of more laboratory periphyton communities to substrate-released Cu generated dose-response relationships and recovery models that were indistinguishable from those generated by the conventional route of exposure. The results of this study demonstrate the utility of chemical-diffusing substrates in field validations of laboratory toxicity tests and in investigations of the effects of stress history on periphyton tolerance to toxicants.