Loss of heterozygosity at the short arm of chromosome 3 occurs frequently in head and neck squamous cell carcinoma (HNSCC). FHIT, a candidate tumor suppressor gene, was recently identified at 3p14.2, and abnormalities of the gene were found in several types of human cancers. To investigate a potential role of the FHIT gene in HNSCC, we examined 16 HNSCC cell lines from 11 patients for abnormalities of the gene by using microsatellite analysis, reverse transcription-PCR, sequencing, and Southern blot analysis. We found that 13 of 16 (81%) cell lines exhibit loss of heterozygosity at 3p14.2. Seven cell lines from six individuals exhibited abnormal transcription patterns, including lack of a FHIT transcript in three lines and shortened transcripts in four lines. A further examination of coding sequences of FHIT in all lines with FHIT transcripts revealed a deletion of exon 4 in one line, a deletion of exons 5 to 7 in one line, and a deletion of exons 5 to 7 plus multiple small insertions between exons 4 and 8 in two lines derived from a primary tumor and a metastasis in the same individual. These results indicate that FHIT may have been inactivated in six cell lines from five (45%) individuals. We also observed two common polymorphism sites at codons 88 and 98 of the gene. These data indicate that abnormal transcription of the FHIT gene is common in HNSCC cell lines; however, other tumor suppressor gene(s) may reside at the same chromosomal region.