Recently, the phosphatase substrate ELF97 phosphate (ELFP) has been employed to study the presence of extracellular phosphatases in different plankton populations in natural aquatic environments. Kinetic properties of ELFP hydrolysis by natural extracellular phosphatases are, however, mostly unknown. We indirectly studied the affinity of extracellular phosphatases for ELFP in different aquatic environments through its ability to inhibit the hydrolysis of 4-methylumbelliferyl phosphate (4MUP). Values of inhibition constants, K_i, which correspond to the concentrations necessary for half saturation of phosphatases by ELFP, were lowest (0.18–4.5 µmol L^–1) in the oligotrophic Mediterranean Sea. We found higher values (i.e. lower affinity) in oligo- to mesotrophic acidified lakes (5.2–14 µmol L^–1), in a eutrophic reservoir (13–35 µmol L^–1) and in a pure culture of the marine bacterium Alteromonas infernus (29 µmol L^–1). ELFP had a pronounced effect on the parameter K_M (Michaelis constant) of 4MUP saturation kinetics, while its effect on the parameter V_max was low. This behaviour is compatible with the assumption of competitive interaction between 4MUP and ELFP. Our experiments indicated that the assay ELFP concentration in the detection kit used was 250–500 µmol L^–1 (after the recommended dilution to a ratio of 1:20), which would ensure >99% saturation of extracellular phosphatases in marine environments and >90% saturation in the studied fresh waters.