The results of a survey aimed at investigating whether NO2 and NH3 emitted by road traffic can influence lichen diversity, lichen vitality and the accumulation of nitrogen in lichen thalli are reported. For this purpose, distance from a highway in a rural environment of central Italy was regarded as the main parameter to check this hypothesis. The results of the present survey indicated that road traffic is not a relevant source of NH3. On the other hand, NO2 concentrations, although rather low, were negatively correlated with distance from the highway according to a typical logarithmic function. No association between NO2 concentrations and the diversity of epiphytic lichens was found, probably because of the low NO2 values measured. Also bark properties were not influenced by distance from the highway. Accumulation of nitrogen, reduction in the content of chlorophyll a, chlorophyll b and total carotenoids were found in transplanted thalli of Evernia prunastri, but NO2 was not responsible for these changes, which were probably caused by applications of N-based fertilizers.