Species-specific extracellular phosphatase activity (PA) was studied in natural phytoplankton with an emphasis on short-term variation in PA and its consequences for the diagnosis of inorganic phosphorus (P) deficiency. Extracellular PA was measured over 3 days at 4-h intervals to compare variations in algal PA in two distinct aquatic environments: in the Rimov reservoir and acidified Plesne Lake. PA was directly detected in an epifluorescence microscope using fluorescently labelled enzyme activity; the fluorescence signal was quantified using image analysis. High short-term variation was found both in the total PA of the samples and specific PA of Ankyra ancora (G. M. Smith) Fott, Monoraphidium dybowski (Wolosz.) Hind. and Kom.-Legn., and Chlorogonium fusiforme Matwienko. The specific PA of these species varied between zero and 157 fmol cell(-1) h(-1). The PA of Chlorogonium fusiforme showed a significant relationship with the time of day. During the study period, populations of the certain species were present that differed in physiological status and activity due to diurnal or random changes caused by mixing in the reservoir or lake, or both. Therefore, species-specific extracellular PA should be interpreted carefully, as an indicator of P deficiency in freshwater phytoplankton if based on limited number of samplings.