Thirty-eight different histidine mutations of Kluyveromyces lactis were isolated and genetically characterized. All of the mutations were nuclear recessive alleles. They turned out to belong to seven different complementation groups, designated hisA1 to hisA7. Five of these genes have been cloned by in vivo complementation of the Klhis mutations. Their homology to some of the histidine genes of Saccharomyces cerevisiae was confirmed by heterologous complementation. However, one of these KlHIS genes did not complement any mutation in the seven known histidine biosynthetic enzymes encoding genes (his1-his7) of S. cerevisiae.