Phytoplasmas are obligate parasites of plants and insects and cause significant crop yield losses worldwide. A number of microar-ray gene expression studies have been performed to understand better the effects of phytoplasma infection on plant physiology. However, little effort has been made for the study of changes in gene expression patterns of the pathogen itself. Quantitative real time PCR in combination with the delta delta Ct method has been shown to be a relatively inexpensive and simple alternative to microarrays. We employed this method to explore whether it is possible to apply this technique for reliable gene expression quan-tification of phytoplasmas on a large scale. In our experimental setup, 242 genes of aster yellows phytoplasma strain witches' broom (AY-WB) were tested for differences in expression in plant and insect host environments, and were shown to be predomi-nantly expressed in the plant or insect hosts. In silico operon prediction corroborated the experimental data. Our findings suggest that the delta delta Ct method can be used to study the physiology of this pathogen.