Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase which plays an important role in the regulation of glutamine catabolism. Antibodies to hepatic glutaminase were used to screen a lambda gt11 rat liver cDNA library. One cDNA to hepatic glutaminase was identified. Changes in the relative abundance of hepatic glutaminase mRNA were determined by hybridization to this cDNA. The mRNA is found only in liver; it is not present prior to birth but its abundance increases dramatically at birth. The abundance of the mRNA is increased approximately 4-fold in diabetes. The sequence of the cDNA was compared to that recently published for kidney (brain)-type glutaminase (Banner, C., Hwang, J.-J., Shapiro, R.A., Wenthold, R.J., Nakatani, Y., Lampel, K.A., Thomas, J.W., Huie, D., and Curthoys, N.P. (1988) Mol. Brain Res. 3, 247-254). When the predicted amino acid sequences were compared a region of 123 amino acids with greater than 80% identity was found. The presence of scattered amino acid substitutions within stretches of identical amino acids suggests that the glutaminase isozymes are encoded by separate genes. This is the first demonstration of any similarity between the two glutaminases at the molecular level.