Quercus species occur widely in East Asia and are major tree species in Japan. The ground-grown seedlings of Quercus serrata and Quercus mongolica var. crispula were exposed to enriched ozone (ambient + 40 ppbv) over one growing season by using open-top-chambers (OTC), and isoprene emissions from the two tree-species were measured across a growing season by using the leaf cuvette method. The isoprene emission rates of the two species were significantly affected by ozone, and the rate of Q. mongolica was reduced to a greater extent by ozone in October than that of Q. serrata. The stomatal conductance of Q. mongolica was higher than that of Q. serrata and not affected by O₃ even in September, suggesting that Q. mongolica absorbed a larger amount of ozone than Q. serrata did. Since the leaf content of the isoprene precursor dimethylallyl diphosphate (DMAPP) was not significantly different between the enriched ozone and filtered air treatments in July and September, isoprene synthase activity in Q. mongolica leaves might have been reduced to a greater extent by the high concentration of ozone due to higher ozone uptake via stomata. Ozone fumigation was also applied to potted Q. serrata seedlings grown in climate chambers to investigate the ozone effect under different growing conditions. The net photosynthetic rate and isoprene emission rate of the potted seedlings were much lower than those of the ground-grown seedlings in the OTC, regardless of the ozone treatments. Between treatments, the isoprene emission rate and DMAPP content were significantly decreased by enriched ozone, suggesting that reduced production of DMAPP is a cause of the decreased isoprene emission. Our two fumigation experiments revealed that the isoprene emission rates of the two Quercus species was decreased by high ozone concentration, but the possible mechanism may not be identical, depending on the experimental conditions.