We have isolated and sequenced a cDNA clone encoding the human gamma enolase. Comparison of our cDNA sequence and the rat gamma enolase sequence revealed 97% homology at the level of amino acid sequence. The two coding regions were 91% homologous on the nucleotide level, whereas the 3' noncoding regions were much less homologous (32%). Further comparison of our cDNA sequence with the human alpha enolase revealed an 82% homology at the amino acid level and a 75% homology at the nucleotide level for the two coding regions, whereas the 3' nontranslated regions were only 30% homologous. Using a portion of the 3' nontranslated region of our cDNA, shown to be specific for human gamma enolase, a single 2.5 kb mRNA was detected in human brain tissue. This same gamma enolase message was also found in a number of human normal nonneuronal tissues, and in several human tumor-derived cell lines. Expression of the mRNA for the gamma enolase subunit should thus be used with caution when identifying the cells of neuronal or neuroendocrine origin.