Stable isotope patterns in lichens are known to vary largely, but effects of substrate on carbon and nitrogen stable isotope signatures of lichens were previously not investigated systematically. N and C contents and stable isotope (δ 15N, δ 13C) patterns have been measured in 92 lichen specimens of Xanthoria parietina from southern Bavaria growing on different substrates (bark and stone). Photobiont and mycobiont were isolated from selected populations and isotopically analyzed. Molecular investigations of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA) region have been conducted on a subset of the specimens of X. parietina. Phylogenetic analysis showed no correlation between the symbionts X. parietina and Trebouxia decolorans and the substrate, isotope composition, or geographic origin. Instead specimens grown on organic substrate significantly differ in isotope values from those on minerogenic substrate. This study documents that the lichens growing on bark use additional or different N sources than the lichens growing on stone. δ 15N variation of X. parietina apparently is controlled predominantly by the mass fraction of the mycobiont and its nitrogen isotope composition. In contrast with mycobionts, photobionts of X. parietina are much more 15N-depleted and show less isotopic variability than mycobionts, probably indicating a mycobiont-independent nitrogen acquisition by uptake of atmospheric ammonia.