Photosynthesis-irradiance (P-I) characteristics of periphyton (microphytobenthos) have been considered primarily for entire assemblages. How P-I responses vary with mat thickness and with community composition has not been considered in detail. We used a combined approach of modeling, microscale determinations of photosynthetic rate and light attenuation, and whole-assemblage O2 flux measurements to explore P-I relationships. The modeling approach suggested that the onset of photosynthetic saturation and photoinhibition will occur at higher irradiance and that whole-mat photoinhibition (decreased photosynthesis at very high irradiance), biomass-specific maximum photosynthetic rate, and initial slope of the P-I function (α) should decrease as assemblage thickness increases or light attenuation increases. Spherical light microsensor profiles for a variety of stream algae indicated a strongly compressed photic zone with attenuation coefficients of 70–1791 m−1 for scalar photosynthetic photon fluence density. The O2 microelectrode measurements showed little if any photoinhibition at 2 and 4 mm depths in one filamentous green algal (Ulothrix) assemblage, with a relatively low attenuation coefficient, and no photoinhibition in a second Ulothrix community. An assemblage dominated by a unicellular cyanobacterium exhibited little photoinhibition at 2 and 4 mm, and a dense cyanobacterial (Phormidium)/xanthophyte (Vaucheria) community exhibited no photoinhibition at all. The microelectrode data revealed increases in α over several millimeters of depth (photoacclimation). These data supported the model predictions with regard to the effects of mat optical thickness on whole-assemblage values for α and photoinhibition. Whole-community O2 flux data from 15 intact assemblages revealed positive relationships between chlorophyll a density and maximum photosynthetic rate or α expressed per unit area; the relationships with chlorophyll a were negative when photosynthetic rates were expressed per unit chlorophyll a. None of the whole assemblages exhibited photoinhibition. Thus, the data from the whole communities were consistent with model predictions.