Photosystem II (PSII) efficiency and CO2 assimilation in response to photon flux density (PFD) and intercellular CO2 concentration (Ci) were monitored simultaneously in leaves of apple, pear, apricot, and cherry with a combined system for measuring chlorophyll fluorescence and gas exchange. When photorespiration was minimized by low O2 (2%) and saturated CO2 (1300 ppm), a linear relationship was found between PSII efficiency and the quantum yield for CO2 assimilation with altering PFD, indicating CO2 assimilation in this case is closely linked to PSII activity. As PFD increased from 80 to 1900 μmol·m–2·s–1 under ambient CO2 (350 ppm) and O2 (21%) conditions, PSII efficiency decreased by increased nonphotochemical quenching and decreased concentration of open PSII reaction centers. The rate of linear electron transport showed a similar response to PFD as CO2 assimilation. As Ci increased from 50 to 1000 ppm under saturating PFD (1000 μmol·m–2·s–1) and ambient O2, PSII efficiency was increased initially by decreased nonphotochemical quenching and increased concentration of open PSII reaction centers and then leveled off with further a rise in Ci. CO2 assimilation reached a plateau at a higher Ci than PSII efficiency because increasing Ci diverted electron flow from O2 reduction to CO2 assimilation by depressing photorespiration. It is concluded that PSII efficiency is regulated by both nonphotochemical quenching and concentration of open PSII reaction centers in response to light and CO2 to meet the requirement for photosynthetic electron transport.