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1. The light : nutrient hypothesis (LNH) states that algal nutrient content is determined by the balance of light and dissolved nutrients available to algae during growth. Light and phosphorus gradients in both laboratory and natural streams were used to examine the relevance of the LNH to stream periphyton. Controlled gradients of light (12–426 μmol photons m−2 s−1) and dissolved reactive phosphorus (DRP, 3–344 μg L−1) were applied experimentally to large flow-through laboratory streams, and natural variability in canopy cover and discharge from a wastewater treatment facility created gradients of light (0.4–35 mol photons m−2 day−1) and DRP (10–1766 μg L−1) in a natural stream. 2. Periphyton phosphorus content was strongly influenced by the light and DRP gradients, ranging from 1.8 to 10.7 μg mg AFDM−1 in the laboratory streams and from 2.3 to 36.9 μg mg AFDM−1 in the natural stream. Phosphorus content decreased with increasing light and increased with increasing water column phosphorus. The simultaneous effects of light and phosphorus were consistent with the LNH that the balance between light and nutrients determines algal nutrient content. 3. In experiments in the laboratory streams, periphyton phosphorus increased hyperbolically with increasing DRP. Uptake then began levelling off around 50 μg L−1. 4. The relationship between periphyton phosphorus and the light : phosphorus ratio was highly nonlinear in both the laboratory and natural streams, with phosphorus content declining sharply with initial increases in the light : phosphorus ratio, then leveling off at higher values of the ratio. 5. Although light and DRP both affected periphyton phosphorus content, the effects of DRP were much stronger than those of light in both the laboratory and natural streams. DRP explained substantially more of the overall variability in periphyton phosphorus than did light, and light effects were evident only at lower phosphorus concentrations (≤25 μg L−1) in the laboratory streams. These results suggest that light has a significant negative effect on the food quality of grazers in streams only under a limited set of conditions. [ABSTRACT FROM AUTHOR]