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A new, simplified protocol for determining particulate adenosine triphosphate (ATP) levels allows for the assessment of microbial biomass distribution in aquatic systems at a high temporal and spatial resolution. A comparison of ATP data with related variables, such as particulate carbon, nitrogen, chlorophyll, and turbidity in pelagic samples, yielded significant and strong correlations in a gradient from the tributaries of the Chesapeake Bay (sigma-t = 8) to the open North Atlantic (sigma-t = 29). Correlations varied between ATP and biomass depending on the microscopic method employed. Despite the much greater effort involved, biomass determined by microscopy correlated poorly with other indicator variables including carbon, nitrogen, and chlorophyll. The ATP values presented here fit well within the range of ATP biomass estimates in the literature for similar environments. A compilation of prior research data from a wide range of marine habitats demonstrated that ATP values can be ranked according to broad trophic gradients, from the deep sea to eutrophic inland waters. Using a mass-based conversion factor of 250, the contribution of biomass to overall particulate organic carbon (POC) ranged from 15% to 30% along the gradient, from the open ocean to locations in the Chesapeake Bay respectively. Our data corroborate the notion that ATP, due to its consistency and simplicity, is a promising high-throughput indicator of cytoplasm volume with distinct benefits over cell counts and measures of chlorophyll or POC.