Ozone is considered to be a major phytotoxic pollutant. It is an oxidizing molecule with harmful effects that can affect human health and vegetation. Due to its phytotoxicity, it constitutes a threat to food security in a context of climate change. Proline accumulation is induced in response to numerous stresses and is assumed to be involved in plant antioxidant defense. We therefore addressed the question of the putative involvement of proline in plant ozone responses by analyzing the responses of two Arabidopsis mutants (obtained in the Col-0 genetic background) altered in proline metabolism and different ecotypes with various degrees of ozone sensitivity, to controlled ozone treatments. Among the mutants, the p5cs1 mutant plants accumulated less proline than the double prodh1xprodh2 (p1p2) mutants. Ozone treatments did not induce accumulation of proline in Col-0 nor in the mutant plants. However, the variation of the photosynthetic parameter Fv/Fm in the p1p2 mutant suggests a positive effect of proline. Proline accumulation induced by ozone was only observed in the most ozone-sensitive ecotypes, Cvi-0 and Ler. Contrary to our expectations, proline accumulation could not be correlated with variations in protein oxidation (carbonylation). On the other hand, flavonols content, measured here, using nondestructive methods, reflected exactly the genotypes ranking according to ozone sensitivity.