We investigated the thermal acclimation of photosynthesis and respiration in black spruce seedlings [Picea mariana (Mill.) B.S.P.] grown at 22/14 °C [low temperature (LT)] or 30/22 °C [high temperature (HT)] day/night temperatures. Net CO2 assimilation rates (Anet) were greater in LT than in HT seedlings below 30 °C, but were greater in HT seedlings above 30 °C. Dark and day respiration rates were similar between treatments at the respective growth temperatures. When respiration was factored out of the photosynthesis response to temperature, the resulting gross CO2 assimilation rates (Agross) was lower in HT than in LT seedlings below 30 °C, but was similar above 30 °C. The reduced Agross of HT seedlings was associated with lower needle nitrogen content, lower ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) maximum carboxylation rates (Vcmax) and lower maximum electron transport rates (Jmax). Growth treatment did not affect Vcmax : Jmax. Modelling of the CO2 response of photosynthesis indicated that LT seedlings at 40 °C might have been limited by heat lability of Rubisco activase, but that in HT seedlings, Rubisco capacity was limiting. In sum, thermal acclimation of Anet was largely caused by reduced respiration and lower nitrogen investments in needles from HT seedlings. At 40 °C, photosynthesis in LT seedlings might be limited by Rubisco activase capacity, while in HT seedlings, acclimation removed this limitation.