The microclimate in facilities for studying effects of elevated CO2 on crops differs from ambient conditions. Open-top chambers (OTCs) increase temperature by 1–3 °C. If temperature and CO2 interact in their effect on crops, this would limit the value of OTC experiments. Furthermore, interaction of CO2 and temperature deserves study because increases in atmospheric CO2 concentration are expected to cause global warming. This paper describes two experiments in which a recently developed cooling system for OTCs was used to analyse the effects of temperature on photosynthesis, growth and yield of spring wheat (Triticum aestivum L., cv. Minaret). Two levels of CO2 were used (350 and 700 ppm), and two levels of temperature, with cooled OTCs being 1.6–2.4 °C colder than noncooled OTCs. Photosynthetic rates were increased by elevated CO2, but no effect of temperature was found. Cross-switching CO2 concentrations as well as determination of A–Ci curves showed that plant photosynthetic capacity after anthesis acclimated to elevated CO2. The acclimation may be related to the effects of CO2 on tissue composition: elevated CO2 decreased leaf nitrogen concentrations and increased sugar content. Calculations of the seasonal mean crop light-use efficiency (LUE) were consistent with the photosynthesis data in that CO2 increased LUE by 20% on average whereas temperature had no effect. Both elevating CO2 and cooling increased grain yield, by an average of 11% and 23%, respectively. CO2 and temperature stimulated yield via different mechanisms: CO2 increased photosynthetic rate, but decreased crop light interception capacity (LAI), whereas cooling increased grain yield by increasing LAI and extending the growing season with 10 days. The effects of CO2 and temperature were not additive: the CO2 effect was about doubled in the noncooled open-top chambers. In most cases, effects on yield were mediated through increased grain density rather than increased individual grain weights. The higher growth response to elevated CO2 in noncooled vs. cooled OTCs shows that a cooling system may remove a bias towards overestimating crop growth response to CO2 in open-top chambers.