The coding sequence for hexokinase enzyme was cloned from Leishmania major. The sequence was found to encode an enzyme with a molecular mass of 51.74 kDa. Amino acid sequence showed maximum homology with known trypanosome and plant hexokinases. It has a calculated isoelectric point of 8.46 and contains an N-terminal peroxisome-targeting signal, the characteristics frequently associated with glycosomal proteins. The sequence indicated the presence of conserved amino acid residues and motifs that are present in plant and mammalian hexokinases; these are apparently involved in the binding of different substrates. The L. major genome was found to have 2 copies of hexokinase coding sequences in tandem with an intergenic spacer of 2.58 kb. Both the genes in the hexokinase locus were transcribed as individual transcripts in a monocistronic form, having the same size as seen by Northern blot analysis. The hexokinase gene was transcribed in large amounts in the promastigote stage, whereas there is only weak expression in the amastigote stage as determined by RT-PCR analysis. Sequencing of hexokinase loci from different Leishmania species (e.g., L. donovani, L. infantum, L. tropica, and L. mexicana) revealed that the hexokinase locus is highly conserved at the DNA and protein levels among species of Leishmania compared with trypanosomes.