Hydrolysis of dissolved organic phosphorus by marine planktonic microorganisms is a key process in the P cycle, particularly in P-depleted, oligotrophic environments. The present study assessed spatiotemporal variations in phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea in two contrasting situations: the end of winter (including a small bloom period) and autumn. The distribution and regulation of the maximum hydrolysis rate (Vm) and half-saturation constant (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached its maximum activities (Vm) after the addition of 1 µM MUF-P (4-methylumbelliferyl phosphate), whereas, for PDE, it was necessary to add up to 50 µM bis(4-methylumbelliferyl)phosphate (bis-MUF-P) to reach saturation state. On average, the Km of PDE was 33 ± 25 times higher than that of PME. The Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm values were on the same order of magnitude for both ectoenzymes, with their ratio (Vm PME : Vm PDE) ranging between 0.2 and 6.3. Dissolved organic phosphorus (DOP) and the phosphomonoesterase hydrolysable fraction of DOP explained most of the lack of variability in Vm PME and Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to the concentration of dissolved inorganic phosphorus. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 µM, high Km, high turnover times) are discussed with respect to the possible unequal distribution of PDE and PME among the size continuum of organic material and accessibility of phosphodiesters.