The amino acid sequence of human hexokinase II was deduced from the sequence of cDNA clones isolated from a skeletal muscle library. An open reading frame of 2751 bases encodes a protein of 917 amino acids. The deduced amino acid sequence has 94% identity with rat hexokinase II but only 72% identity with human hexokinase type I. In addition to hexokinase II clones, the human skeletal muscle cDNA library contained at least an equal number of clones of hexokinase I, the isoform reported to be typically found in kidney and brain. Genetic variation in hexokinase II could underlie insulin resistance in peripheral tissues and cause non-insulin-dependent diabetes mellitus. The availability of this sequence would facilitate investigating the role of mutations in the HKII gene in the etiology of this disease.