Elongation factor-1 (EF-1) gamma is overexpressed in a high proportion of gastrointestinal cancers. The mechanism of overexpression has not been determined. The purpose of this study was to examine cDNA specimens from pancreatic and colorectal cancer for mutation in this gene, which would allow us to determine whether gene mutations are responsible for overexpression of EF-1gamma. In one colorectal carcinoma, we detected an A-->G transition at amino-acid codon 158 (T-->C in the sense strand) resulting in a change from a leucine to a serine. The base change was not detected in cDNA isolated from normal-appearing tissue from the same patient. We did not find mutations in another five colorectal carcinoma and five pancreatic cancer samples. Thus, although we detected a mutation in one tumor, the frequency of mutations was too low to account for the high frequency of overexpression of the EF-1gamma RNA in colorectal cancer. We also investigated other possible mechanisms of overexpression of the EF-1gamma RNA in this study. Slot-blot analysis of DNA isolated from colorectal cancers showed that the overexpression was not due to gene amplification. Using serum starvation to synchronize cultured cells, we showed that the overexpression was also not due to an increase in the number of cycling cells, as occurs in cancer. Using Southern blot analysis, we were unable to detect genome rearrangements that could have been responsible for the overexpression. In conclusion, the mechanism for overexpression of the EF-1gamma gene in colorectal and pancreatic cancer remains unknown. However, mutations in the coding sequence of the gene, gene amplification, and gene rearrangement do not account for the high frequency of overexpression of this gene, and the overexpression is not due to an increase in the number of cycling cells.