Comfrey (Symphytum Officinale) has been used as a vegetable and herbal remedy for many years. It, however, is a mutagen and carcinogen in liver and a possible genotoxin in lung. In order to evaluate the mutagenicity of comfrey and the mechanisms of action in rat lung, we examined the mutant frequencies (MF) and mutational types in the lung cII gene from transgenic Big Blue rats treated with 8% comfrey for 12 weeks. The CII MF in the lungs for rats fed with comfrey was 48 ± 9 × 10-6, which was significantly greater than the MF for control rats, 34 ± 10 × 10-6 (p = 0.026). The mutational spectrum in lung from comfrey-fed rats is significantly different from that in the control. G:C → T:A transversion (29%) was the major type of mutation in comfrey-fed rats, whereas G:C → A:T transition (63%) was the predominant mutation in the controls. An unusual type of tandem-base substitution (4%) was observed among the mutations from comfrey-fed rats that were not seen in the control. This type of tandem-base substitution has been suggested as a mutational signature for the genetic damage of pyrrolizidine alkaloids (PAs). The results indicate that comfrey is mutagenic in rat lung and the types of mutations induced by comfrey are similar to those by other PAs; and suggest that the mutagenicity of comfrey in rat lung results from the genotoxicity of PAs in the plant.