Phosphorus (P) availability drives niche differentiation in the most abundant phytoplankter in the oceans, the marine cyanobacterium Prochlorococcus. We compared the molecular response of Prochlorococcus strain MED4 to P starvation in batch culture to P-limited growth in chemostat culture. We also identified an outer membrane porin, PMM0709, which may allow transport of organic phosphorous compounds, rather than phosphate as previously suggested. The expression of three P uptake genes, pstS, the high-affinity phosphate-binding component of the phosphate transporter, phoA, an alkaline phosphatase, and porin PMM0709, were strongly upregulated (between 10- and 700-fold) under both P starvation and limitation. pstS exhibits high basal expression under P-replete conditions and is likely necessary for P uptake regardless of P availability. A P-stress regulatory gene, ptrA, was upregulated in response to both P starvation and limitation although a second regulatory gene, phoB, was not. Elevated expression levels (>10-fold) of phoR, a P-sensing histidine kinase, were only observed under conditions of P limitation. We suggest Prochlorococcus in P-limited systems are physiologically distinct from cells subjected to abrupt P depletion. Detection of expression of both pstS and phoR in field populations will enable discernment of the present P status of Prochlorococcus in the oligotrophic oceans.