Seasonal changes in size-fractionated alkaline phosphatase activity (APA) were studied in the hypereutrophic Villerest reservoir (Roanne, France) to determine whether the APA showed seasonal variations coupled to abiotic and biotic variables. Samples were collected at different depths from November 1992 to October 1993. The phosphatase activities were measured spectrophotometrically with pnitrophenyl phosphate as the substrate and related to protein concentrations. No correlations were found between APA and soluble reactive phosphorus concentrations during an annual cycle. Free phosphatases were particularly high in the epilimnion (54.5% of total activity was at 5 m) and decreased with depth (4% of total APA at 40 m). The contribution of small microorganisms to APA increased with depth. It was concluded that APA is not a valid quantitative test of the phosphorus status of aquatic microorganisms.