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Lipid content and lipid class composition were determined in stream periphyton and the filamentous green algae Cladophora sp. and Spirogyra sp, Sterols and phospholipids were compared to chlorophyll a (chl a) as predictors of biomass for stream periphyton and algae. Chlorophyll a, phospholipids, and sterols were each highly correlated with ash‐free dry mass (AFDM) (r2 > 0.98). Stream periphyton exposed naturally to high light (HL) and low light (LL) had chl a concentrations (μg chl a‐mg−1AFDM) of 7.9± 0.7 and 12.4 ± 2.9, respectively, while the sterol concentrations of these HL and LL stream periphyton (1.6 ± 0.4) were not significantly different (P > 0.05). Periphyton exposed to an irradiance of 300 μmol photons·m−2s−1 in the laboratory for 60 h had 5.6 ± 0.55 μg chl a·mg−1 AFDM, but the same periphyton exposed to 2% incident light for the same amount of time had 11.0 ± 0.56 μg chl a·mg−1 AFDM. Sterol concentrations in these periphyton communities remained unchanged (1.5 ± 0.3 μg·mg−1AFDM), Similar results (i.e. changes in chl a but stability of sterol concentrations in response to irradiance changes) were also found for Cladophora and Spirogyra in laboratory experiments. Sterols can be quantified rapidly from a few milligrams of algae and appear to be a useful predictor of eukaryote biomass, whereas cellular levels of chl a vary substantially with light conditions. Phospholipids (or phospholipid fatty acids) are considered to be a reliable measure of viable microbial biomass. Nevertheless, phospholipid content varied substantially and unpredictably among algae and periphyton under different light regimes. Irradiance also had a significant effect on storage lipids: HL Cladophora and HL periphyton had 2 × and 5 × greater concentrations of triacylglycerols, respectively, compared to their LL forms. HL and LL algae also differed in the concentration of several major fatty acids. These light‐induced changes in algal lipids and fatty acids have important implications for grazers.

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