Japanese flounder Paralichthys olivaceus glyceraldehyde-3-phosphate dehydrogenase (GAPDH or G3PD) cDNA was cloned and sequenced. Japanese flounder GAPDH was found to consist of 333 amino acid residues and to exhibit a high degree of homology to previously reported GAPDH of vertebrates, in both the DNA and amino acid sequences. Curiously, Japanese flounder GAPDH was found to be more homologous to GAPDH of Xenopus, chicken and mammals than to that of rainbow trout. We developed primers for RT-PCR and used this method to amplify GAPDH mRNA from various tissues to determine whether it could be used as an internal standard. The GAPDH was expressed in all organs and tissues, and confirmed that it represents a good internal standard positive control for the study of gene expression.