The relative contribution of promoter hypermethylation and aberrant splicing to the inactivation of the fragile histidine triad (FHIT) gene is unclear. Using genetic and epigenetic analyses, the current investigation examines the loss of protein and mRNA expression, and 5'CpG hypermethylation and allelic imbalance of the FHIT gene in a series of 129 non-small cell lung cancer (NSCLC) samples, in parallel with clinicopathological analyses. We found that 50% of NSCLC patients had aberrant protein expression, which was more frequent in squamous cell carcinomas (SQ) (69%) than in adenocarcinomas (AD) (28%) (P < 0.0001). 5'CpG hypermethylation of FHIT was identified in 31% of patients. Abnormally-sized FHIT transcripts were also observed in 24% of patients and were attributed to various exonic deletions, mainly in the region of exons 4-8. Allelic imbalance of the FHIT locus and its correlation with the status of Fhit expression, 5'CpG hypermethylation, and aberrant splicing, indicated that biallelic inactivation of Fhit expression could be induced by 5'CpG hypermethylation of one allele and alternative splicing in the other allele. Moreover, an 83% concordance in the methylation status of FHIT was demonstrated between 12 samples of bronchial precancerous lesions taken before surgery and their matched resected tumours. Our data suggest that FHIT 5'CpG hypermethylation and splicing alterations are both predominant mechanisms involved in the aberrant expression of the FHIT gene, and that FHIT 5'CpG methylation may be potentially used as a supplemental detection marker for NSCLC.