Imbalance of the Ras signaling pathway is a major hallmark of human cancer. In this context, activating point mutations of the K-ras oncogene are a common feature of many tumor types. The discovery of methylation-mediated silencing of the Ras-effector homologue RASSF1A has revealed another way by which this cellular pathway may be altered. Inactivation by hypermethylation of a RASSF1A homologue, NORE1A, has recently been observed in human cancers. If both K-ras and NORE1A act in the same pathway, simultaneous molecular lesions in the two genes in the same tumor should be a rare event. To test whether this inverse association exists, we have analysed the K-ras mutational status and NORE1A CpG island hypermethylation of 61 non-small-cell lung carcinomas and the methylation status of the two other Ras effectors, RASSF1A and HRASLS. No association was found between the methylation status of NORE1A, RASSF1A and HRASLS or the status of K-ras with respect to the latter two genes. However, our results demonstrate that the epigenetic alteration of NORE1A is confined to lung tumors with a wild-type K-ras: 88% (15 of 17) of the tumors with NORE1 hypermethylation did not harbor a K-ras mutation (P=0.008, Fisher's exact test). Thus, the mutual exclusivity of the epigenetic and genetic alterations in the two genes of the Ras pathway suggests that they play a critical and cooperative role in human tumorigenesis.