To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-rastmΔ4A/− mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras+/− mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-rastmΔ4A/tmΔ4A mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-rastmΔ4A/tmΔ4A mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras+/− and K-rastmΔ4A/− mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.