Measurement of algal photosynthetic performance with conventional methods requires thousands of cells obtained by isolation and subsequent cultivation. This is a time-consuming process for many species. We describe a new method to study photosynthetic performance of single algal cells under various environmental conditions by a combination of modulated chlorophyll fluorescence, light microscopy, and sample manipulation techniques. Single cell fluorescence was measured with a modulated microfluorometer integrated in an inverted microscope. The algal cell was sucked onto the tip of a glass microcapillary and positioned in the center of the field of view of the microscope by a micromanipulator. A superfusion device was used to generate a flow of experimental solution of variable composition along the alga. The light dependence of Scenedesmus obtusiusculus single-cell photosystem II (PSII) electron flow was measured at various pH. At a high light intensity PSII electron flow was inhibited at pH 6.5 and higher, while at a low light inhibition occurred at pH 9.5. This is in agreement with inhibition of photosynthesis by substrate (CO2) limitation at alkaline pH. This approach can easily be extended to study the in vivo effects of other abiotic parameters (temperature, nutrients, toxicants, oxygen) on the photosynthetic performance of algae.