Effects of substratum and water nutrient perturbations on periphyton growth were assessed in a nutrient-poor stream by combining a substratum enrichment technique with a flow-through bioassay system. Periphyton growth (chlorophyll a, total biovolume) responded to combined influences of water and substratum enrichment in an additive manner when both compartments were amended with N and P to yield an optimal ratio (N:P = 16:1). When NO3-N was added to the substratum and PO4-P to the water, algal growth response was synergistic. Analysis of the vertical distribution of P fractions in cores taken from nutrient-diffusing substrata indicates that attached microorganisms mediate P release from underlying substrata, acting as a filter or temporary sink. Nutrient-diffusing substrata are useful detectors of limiting nutrients in aquatic systems; however, their function and application differ from water enrichment assays where nutrients are added at a constant rate. Differences are partially attributed to spatial and temporal variability of nutrient release and the strictly localized influence of substratum flora on ambient water chemistry.