The photosynthetic performance of an epilithic cyanobacterial biofilm was studied in relation to the in situ light field by the use of combined microsensor measurements of O₂, photosynthesis, and spectral scalar irradiance. The high density of the dominant filamentous cyanobacteria (Oscillatoria sp.) embedded in a matrix of exopolymers and bacteria resulted in a photic zone of <0.7 mm. At the biofilm surface, the prevailing irradiance and spectral composition were significantly different from the incident light. Multiple scattering led to an intensity maximum for photic light (400-700 nm) of ca. 120% of incident quantum irradiance at the biofilm surface. At the bottom of the euphotic zone in the biofilm, light was attenuated strongly to <5-10% of the incident surface irradiance. Strong spectral signals from chlorophyll a (440 and 675 nm) and phycobilins (phycoerythrin 540-570 nm, phycocyanin 615-625 nm) were observed as distinct maxima in the scalar irradiance attenuation spectra in the upper 0.0-0.5 mm of the biofilm. The action spectrum for photosynthesis in the cyanobacterial layer revealed peak photosynthetic activity at absorption wavelengths of phycobilins, whereas only low photosynthesis rates were induced by light absorption of carotenoids (450-550 nm). Respiration rates in light- and dark-incubated biofilms were determined using simple flux calculations on measured O₂ concentration profiles and photosynthetic rates. A significantly higher a real O₂ consumption was found in illuminated biofilms than in dark-incubated biofilms. Although photorespiration accounted for part of the increase, the enhanced areal O₂ consumption of illuminated biofilms could also be ascribed to a deeper oxygen penetration in light as well as an enhanced volumetric O₂ respiration in and below the photic zone. Gross photosynthesis was largely unaffected by increasing flow velocities, whereas the O₂ flux out of the photic zone, that is, net photosynthesis, increased with flow velocity. Consequently, the amount of produced O₂ consumed within the biofilm decreased with increasing flow velocity. Our data indicated a close coupling of photosynthesis and respiration in biofilms, where the dissolved inorganic carbon requirement of the photosynthetic population may largely be covered by the respiration of closely associated populations of heterotrophic bacteria consuming a significant part of the photosynthetically produced oxygen and organic carbon.