A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.