We compared inorganic phosphate (Pi) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate-limited cultures had up to six times higher maximum Pi uptake rates than P-replete cultures in both strains. For strain GBRTRLI101, cell-specific Pi uptake rates were nearly twice as high, due to larger cell size, but P-specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 μM for Pi uptake and 0.1 and 0.2 μM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing Pi up to 1.0 μM. The C:P ratios were 340–390 and N:P ratios were 40–45 for both strains under severely P-limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 μM Pi. The North Atlantic strain IMS101 is better adapted to growing on Pi at low concentrations than is GBRTRLI101 from the more Pi-enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) Pi concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.