A new 33 P-tracer technique was used to define low-level phosphate (Pi) uptake kinetics for a dominant tropical seagrass, Thalassia testudinum. We established seagrass Pi uptake kinetics at high (western Bay) and low (eastern Bay) nutrient sites in a fine-grained carbonate lagoon of south Florida (Florida Bay), where P limitation of seagrass has been documented. Sediment Pi adsorption kinetics were also investigated to test whether carbonate sediments could sequester Pi to the threshold levels we established for T. testudinum, defined as S min or the physiological threshold value where Pi uptake is positive. At Pi levels characteristic of Florida Bay (#0.26 mmol L 21 ), leaf and root Pi uptake was linear with increasing levels of Pi, and similar affinities (a) were found for leaves and roots (0.12‐0.30 and 0.10‐0.20 mmol g dry weight 21 h 21 ). Smin for roots and leaves was extremely low (0.004‐0.009 mmol L 21 ) regardless of the P status of the site. While T. testudinum was able to take up Pi at nanomolar levels, the uptake rates were insufficient for plant requirements. Thus, sediment pools or transient fluxes of P to the water column must sustain high seagrass production rates in the Bay. Sediment adsorption‐desorption equilibrium for Pi was 10-fold lower in the eastern versus western Bay sites, meeting ,10% and .87% of the P demand for T. testudinum, respectively, a result that might account for the reported P limitation of seagrass biomass and production in eastern Florida Bay.