The regulation of large rivers to meet human requirements (e.g. hydroelectricity production, flood prevention, recreation activities) alters the longitudinal distribution of plankton communities and may affect their capacity to use nutrients and organic matter. Here we analyzed phosphorus (P) availability and use by phytoplankton and bacterioplankton in 6 upstream and 5 downstream sites from a reservoir system in the Ebro River (N Spain). Alkaline phosphatase activity (APA) was related to nutrient availability and biomass of both phytoplankton and bacterioplankton. During dry periods phytoplankton and bacterioplankton APA was inversely correlated to P availability in the water, but these patterns became less clear during wet periods. The phosphorus-APA patterns were more consistent in the upstream sites and especially during dry periods. Although phytoplankton APA was 6-40 times greater than that of bacterioplankton, APA per unit of biomass suggested that bacterioplankton was more efficient at utilizing dissolved organic phosphorus (DOP) in the upstream section during dry periods. Imbalanced N:P ratios in the particulate (N:P ranging 133-170) and dissolved (N:P ranging 301-819) water fractions confirmed the strong P limitation in these upstream communities. The phosphorus-APA patterns were weaker in the downstream section and during wet periods. The reservoirs caused a change in the downstream dynamics, where bacterioplankton biomass was positively correlated to APA but APA per unit of biomass decreased. Our findings reveal that river regulation drives changes in plankton use of organic phosphorus, especially during extreme dry periods.