It is expected that climate change will expand the open oligotrophic oceans by enhanced thermal stratification. Because temperature defines the geographic distribution of picophytoplankton in open-ocean ecosystems and regulates photophysiological responses, it is important to understand how temperature affects picophytoplankton growth and photophysiology. Two prokaryotic and 2 eukaryotic picophytoplankton strains were acclimated to 3 different temperatures, ranging from 16 to 24 degrees C. Temperature-dependent growth and photophysiology were assessed by measurements of specific growth rates, cell size, pigment composition, absorption and electron transport rates. Growth of Prochlorococcus marinus (eMED4), Prochlorococcus sp. (eMIT9313), Ostreococcus sp. (clade B) and Pelagomonas calceolata was positively related to temperature, especially in the prokaryotic strains. Changes in photophysiology included increased light harvesting, increased electron transport and reduced photoinhibition at elevated temperatures. However, the changes related to light harvesting and electron transport could not fully explain the observed difference in growth. This suggests that other processes, such as Calvin cycle activity, are likely to limit growth at sub-optimal temperatures in these picophytoplankton strains. The overall changes in photophysiology during temperature acclimation will possibly allow photosynthesis at higher irradiance intensities, but the genetically defined low temperature tolerances and photosynthetic characteristics of the different ecotypes will likely be more important in determining picophytoplankton (depth) distribution and community composition.