The substrate [α‐32P]ATP was used to represent the available fraction of the dissolved phosphomonoester (PME) pool and to measure its rate of assimilation by freshwater plankton in systems of low phosphorus availability. We compared [32P]ATP uptake to inorganic 32PO43‒ uptake. The upper bounds to the pool sizes of PO43‒ and PME were estimated with Rigler bioassays, and these values, together with the uptake constants at ambient substrate concentrations, permit comparison of the uptake of organic and inorganic P by limnetic plankton. Affinity of the plankton for both substrates was high, and the estimated flux rates from both pools were similar, demonstrating that PME contributes significantly to total P uptake. We tested the hypothesis that algae >1 µm preferentially assimilate P from PME but observed that the 0.2–1‐µm size fraction dominates uptake of P from either source at ambient concentrations. The specific flux of P from PME into the 1–12‐µm size fraction was often as high as that into the 0.2–1‐µm fraction, but plankton >12 µm typically incorporate <5% of the P from either source.