The evolution of the diversity of plant secondary metabolites is still poorly understood. To determine whether natural enemies could exert selection on plant secondary chemistry, pathogen infestation and invertebrate herbivory were measured on 10 genotypes of Senecio jacobaea (Tansy Ragwort) at two experimental field sites during a 2-year period. The genotypes represented two chemotypes based on the presence of the pyrrolizidine alkaloids (PA) jacobine and erucifoline. At one site, Heteren, mainly generalist herbivores were present. Here, damage was limited and did not differ among genotypes or chemotypes. At the other site, Meijendel, several specialists attacked the plants. Damage increased during the year, with a peak in July when most damage was caused by the specialist moth Tyria jacobaeae. At this peak there was no difference in damage among chemotypes. In the months prior to T. jacobaeae damage, chemotypes with jacobine were more severely attacked by specialists than the chemotypes without jacobine. Total damage during that period was positively correlated with both total PA concentration and jacobine concentration. Probably plant vigor also played a role in host preference since damage per individual plant was positively correlated with plant size. Our results suggest that total PA concentration and specifically jacobine had a positive effect on specialist feeding, indicating ecological costs involved in the production of PAs. Ecological costs related to plant secondary compounds could explain why not all individuals produce high levels of these compounds. In addition, differences in specialist herbivore pressures among sites may contribute to the variation in secondary metabolites among populations.