High bulk extracellular phosphatase activity (PA) suggested severe phosphorus (P) deficiency in plankton of three acidified mountain lakes in the Bohemian Forest. Bioavailability of P substantially differed among the lakes due to differences in their P loading, as well as in concentrations of aluminum (Al) and its species, and was accompanied by species-specific responses of phytoplankton. We combined the fluorescently labeled enzyme activity (FLEA) assay with image cytometry to measure cell-specific PA in natural populations of three dinophyte species, occurring in all the lakes throughout May–September 2007. The mean cell-specific PA varied among the lakes within one order of magnitude: 188–1,831 fmol · cell−1 · h−1 for Gymnodinium uberrimum (G. F. Allman) Kof. et Swezy, 21–150 fmol · cell−1 · h−1 for Gymnodinium sp., and 22–365 fmol · cell−1 · h−1 for Peridinium umbonatum F. Stein. To better compare cell-specific PA among the species of different size, the values were normalized per unit of cell biovolume (amol · μm−3 · h−1) for further statistical analysis. A step-forward selection identified concentrations of total and ionic Al together with pH as significant factors (P < 0.05, Monte Carlo permutation test), explaining cumulatively 57% of the total variability in cell-specific PA. However, this cell-specific PA showed an unexpected reverse trend compared to an overall gradient in P deficiency of the lake plankton. The autecological insight into dinophyte cell-specific PA therefore suggested other factors, such as light availability, mixotrophy, and/or zooplankton grazing, causing further PA variations among the acidified lakes.