Picophytoplankton account for most of the marine (sub-)tropical phytoplankton biomass and primary productivity. The contribution to biomass among plankton functional types (PFTs) could shift with climate warming, in part as a result of different physiological responses to temperature. To model these responses, Eppley's empirical relationships have been well established. However, they have not yet been statistically validated for individual PFTs. Here, we examine the physiological response of nine strains of picophytoplankton to temperature; three strains of picoprokaryotes and six strains of picoeukaryotes. We conduct laboratory experiments at 13 temperatures between -0.5 and 33 • C and measure the maximum growth rates and the chlorophyll a to carbon ratios. We then statistically validate two hypotheses formulated by Eppley in 1972: The response of maximum growth rates to temperature (1) of individual strains can be represented by an optimum function, and (2) of the whole phytoplankton group can be represented by an exponential function Eppley (1972). We also quantify the temperature-related parameters. We find that the temperature span at which growth is positive is more constrained for picoprokaryotes (13.7-27 • C), than for picoeukaryotes (2.8-32.4 • C). However, the modeled temperature tolerance range ( T) follows an unimodal function of cell size for the strains examined here. Thus, the temperature tolerance range may act in conjunction with the maximum growth rate to explain the picophytoplankton community size structure in correlation with ocean temperature. The maximum growth rates obtained by a 99th quantile regression for the group of picophytoplankton or picoprokaryotes are generally lower than the rates estimated by Eppley. However, we find temperature-dependencies (Q 10 ) of 2.3 and of 4.9 for the two groups, respectively. Both of these values are higher than the Q 10 of 1.88 estimated by Eppley and could have substantial influence on the biomass distribution in models, in particular if picoprokaryotes were considered an independent PFT. We also quantify the increase of the chlorophyll a to carbon ratios with increasing temperature due to acclimation. These parameters provide essential and validated physiological information to explore the response of marine ecosystems to a warming climate using ocean biogeochemistry models.