Changing environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen-activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O3) to generate ROS in the apoplast of wild-type Col-0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full-level AtMPK3 activation by O3. In addition, we show that AtMPK3, but not AtMPK6, responded to O3 transcriptionally and translationally during O3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O3 treatment. The use of O3 to induce apoplastic ROS formation offers a non-invasive in planta system amenable to reverse genetics that can be used for the study of stress-responsive MAPK signaling in plants.