Two glutamate dehydrogenase (GDH) cDNA sequences encoding 474 (GDH A, a truncated gene) and 552 amino acids (GDH B) were discovered in the Pacific white shrimp, Litopenaeus vannamei. Both GDH A and GDH B were fairly conserved as shown by the amino acid alignment. The two cDNAs differed only at the C-terminal sequences. The quantitative real time polymerase chain reaction (qPCR) assay was developed to estimate the specific expression on different tissues. Both GDH A and B were mainly expressed in shrimp muscle, followed by in the gill and epithelium, while the expression in shrimp hepatopancreas was the lowest when compared with other tissues. Though no significant difference was observed among all the test tissues, the ratio of GDH B to GDH A transcription was kept at a very high level ranging from 32.04 in the eyestalk to 64.52 in the muscle, indicating that GDH B was largely expressed in L.vannamei. The GDH activity in the muscle of L.vannamei was also the highest followed by in the epithelium, eyestalk and gill. The GDH activity in hepatopancreas was significantly lower than that in other tissues. As a control, the tissue expression of Na -K ATPase was also determined. This enzyme was mainly expressed in the gill, followed by in the muscle and epithelium. Our results provide baseline data in physiological expressions of GDH and Na -K ATPase for osmoregulation in L.vannamei. The research protocol offers a practical guideline for the analysis of gene expression in shrimp. The sequences and techniques developed in this study will also contribute to the GDH gene research in other crustacean species.