We examined 2 normal pancreata, 21 primary pancreatic ductal cancers, and 19 pancreatic cancer cell lines for Fhit expression and FHIT gene status. The normal pancreas expressed Fhit protein in the cytoplasm of ductular cells, whereas interlobular and larger ducts, acini, and insulae of Langerhans were negative. Fhit protein was detected by immunoblot assay in 11 pancreatic cancer cell lines; of the 8 cell lines lacking Fhit protein, 7 lacked FHIT mRNA and 1 showed an abnormally sized transcript. DNA from five of these eight cell lines showed homozygous loss of FHIT exon 5. In 8 of the 21 primary cancers, Fhit expression was detected by immunohistochemistry. Reverse transcription-PCR analysis of 6 of the 13 cases lacking Fhit showed normal-sized FHIT product in 3 cases and a mixture of normal and abnormal products in the other 3. Sequencing showed that abnormal bands were missing variable numbers of exons. Loss of microsatellite DNA markers internal to the FHIT gene was observed in 10 of 13 primary cancers lacking Fhit protein (homozygous in two cases) and in only 1 of the 8 cancers expressing Fhit protein. In nine primary cancers, four expressing and five lacking Fhit protein, it was possible to obtain pure cancer DNA by microdissection. Three of the five microdissected cases lacking Fhit protein exhibited homozygous deletion of FHIT exon 5. In conclusion, the lack of Fhit protein in pancreatic cancers correlated with absence or alteration of FHIT mRNA and was often associated with FHIT gene anomalies.