Intra-genus and intra-specific variation and the influence of nitrogen enrichment on net assimilation and stomatal conductance of some annual Trifolium species of Mediterranean dehesa grasslands were assessed under experimental conditions. Also gas exchange rates were compared between some Leguminosae and Poaceae species growing in the field in a dehesa ecosystem in central Spain. The results showed that the previously reported different O3 sensitivity of some Trifolium species growing in pots does not seem to be related to different maximum g(s) values. In addition, no clear differences on gas exchange rates could be attributed to Leguminosae and Poaceae families growing in the field, with intra-genus variation being more important than differences found between families. Further studies are needed to increase the database for developing a flux-based approach for setting O3 critical levels for semi-natural Mediterranean species.