Phosphate is an important nutrient that restricts microbial production in many freshwater and marine environments. The actual concentration of phosphate in phosphorus-limited waters is largely unknown because commonly used chemical and radiochemical techniques overestimate the concentration. Here, using a new steady-state radiobioassay to survey a diverse set of lakes, we report phosphate concentrations in lakes that are orders of magnitude lower than estimates made spectrophotometrically or with the frequently used Rigler radiobioassay. Our results, combined with those from the literature, indicate that microbes can achieve rapid turnover rates at picomolar nutrient concentrations. This occurs even though these concentrations are about two orders of magnitude below the level where phosphate uptake is estimated to be half the saturation level for the pico-plankton community. Also, while phosphate concentration increased with the concentration of total phosphorus and soluble reactive phosphorus in the lakes we sampled, the proportion of phosphate in the total phosphorus pool decreased from oligotrophic to eutrophic lakes. Such information, as revealed by the phosphate assay that we use here, should allow us to address hypotheses concerning the concentration of phosphate available to planktonic microorganisms in aquatic systems.