Michaelis–Menten kinetics were determined for alkaline phosphatase, aminopeptidase and β-glucosidase in the Mediterranean Sea. Although the ectoenzymatic-hydrolysis contribution to heterotrophic prokaryotic needs was high in terms of N, it was low in terms of C. This study points out the biases in interpretation of the relative differences in activities among the three tested enzymes in regard to the choice of added concentrations of fluorogenic substrates.