Although only a small proportion of plant phosphorus (P) is used for photosynthesis, the relationships between P and photosynthesis can be strong. It was hypothesized, in this study, that variation in the allocation of orthophosphate (Pi) between active (cytoplasmic) and nonactive (vacuolar) pools would underpin differences in rates of photosynthesis in 4-month-old Eucalyptus globulus seedlings grown with a varying P supply. Photosynthetic biochemistry was assessed by the response of net photosynthesis to increasing intercellular [CO2]. Cytoplasmic Pi was sequestered as mannose 6-phosphate. Total P and the proportion of P as Pi were positively related to P supply. The ratios of active : stored Pi (10-24%) varied little over the range of treatments. Active Pi was positively related to P supply, as was photosynthesis (7 micromol CO2 m(-2) s(-1) with 0 mM P vs. 16 micromol CO2 m(-2) s(-1) with 0.32 mM P). Positive relationships between P supply and photosynthesis were explained best by leaf P content, not by active pools of Pi. The distribution of Pi between the vacuole and the cytoplasm had little impact on the photosynthetic phosphorus-use efficiency (PPUE), and reductions in cytoplasmic Pi had little effect on photosynthesis. Hence, PPUE is an unsuitable guide for assessing plant responses to increasingly unavailable P in the environment.