Glycerate kinase activity was less than 5% of normal, whereas D-glycerate dehydrogenase (glyoxylate reductase) and triokinase activities were normal. The longer variant, GLYCTK1, encodes a deduced 523-amino acid protein. The shorter variant, GLYCTK2, which lacks exon 4, encodes a deduced 234-amino acid protein. PCR analysis of 17 human tissues detected variable levels of both transcripts in all tissues examined. In situ hybridization of adult mouse tissues showed Glyctk in several specific brain regions, in renal cortex interstitial cells, in epithelium of the interlobular bile duct, and in skeletal muscle. Fluorescence-tagged GLYCTK1 and GLYCTK2 were localized in cytoplasmic particles in transfected COS-7 cells. GLYCTK2, but not GLYCTK1, localized in mitochondria.