Ramularia collo-cygni, a pathogenic deuteromycete, is the causal agent of Ramularia leaf spot disease on barley. It produces a number of coloured metabolites of which one was identified as rubellin D, a phytotoxin only described for Mycosphaerella rubella until now. Rubellin D induces light- and concentration-dependent necrosis on barley leaves. In a biochemical model system we could demonstrate that rubellin D exhibits photodynamic activity triggering the light-dependent production of reactive oxygen species (ROS). The use of inhibitors of ROS production confirmed mainly the production of singlet oxygen and to a lesser extent superoxide. In addition, light-dependent peroxidation of α-linolenic acid by rubellin D was shown. Based on our results we present a model for ROS-mediated symptom induction on barley leaves by Ramularia collo-cygni.