Photosynthetic organisms possess a great diversity of mechanisms to protect themselves from the potentially stressful effects of high PAR (photosynthetically active radiation). A distinctive response to longer term exposure to high levels of PAR in lichens is the synthesis of a variety of substances in the upper cortex that can protect photobionts from photoinhibition. In the present study, lichen substances were removed harmlessly from lichens using the “acetone rinsing” method. This enabled us to compare the importance of the substances in photoprotection in sun and shade collections of four species of Afromontane lichens. While all species normally grow in more exposed microhabitats, it is easy to make collections of more shaded thalli. Using chlorophyll fluorescence, we show that collections of lichens from sunny microhabitats have higher tolerance to photoinhibition than those from shaded locations. Furthermore, removal of lichen substances increases sensitivity to photoinhibition, suggesting that even although colorless, they have a role in protecting against high PAR. Sensitivity was increased much more in sun than shade collections, implying that substances play a greater role in photoprotection in lichens from sunny microhabitats. Nevertheless, following the removal of lichen substances, most sun collections still possess higher tolerance to photoinhibition than shade collections. Therefore, the additional tolerance of sun collections appears derive from a combination of both lichen substances and other, probably more biochemical tolerance mechanisms.