Regulation of large rivers for human purposes (e.g. hydroelectricity production, flood prevention, recreation activities) may broken down the longitudinal patterns on microbial planktonic communities, altering their role in organic matter production and nutrient recycling in river ecosystems. The present study aims to analyse the relationships between phosphorus nutrient sources and phosphatase activity in the particulated and dissolved water fractions of the Ebro river (NE Spain). Whit this purpose, we measured the nutrient content and phosphatase activity from the two water fractions in 6 sites upstream and 6 downstream of a reservoirs system during three years. Algal and bacterial densities and phytoplankton community composition were analysed throughout the study. Our working hypothesis was that the temporal patterns (seasonality) would mainly determine the phosphatase activity between water fractions, though spatial differences between river sections (upstream and downstream of reservoirs) may interact in its regulation. Results showed higher phosphatase activity in spring-summer than in autumn-winter which agreed to the increases in plankton cell densities along the two sections. It was during spring-summer, when strong correlation between water SRP and phosphatase activity in the two water fractions were observed. Instead, these trends shifted during autumn-winter months when SRP-phosphatase correlations were only consistent in the dissolved fraction. Irrespectively of the study period, relationships between SRP-phosphatase in the upstream section were stronger in the particulated fraction while in the downstream section stronger for the dissolved fraction. These differences could be explained by the differences in microbial cell densities and phytoplankton species composition found between sections. The present research shows that phosphatase activity patterns in large regulated rivers did not follow a progressive increase to the lower sections. The uncoupling between nutrients and phosphatase activity in the particulated fraction (phytoplankton dominated) might evolve on a decrease in authochthonous organic matter production in lower water courses.