Candida albicans possesses a cyanide-resistant respiratory pathway mediated by alternative oxidase (AOX), which seems to be encoded by a gene family with two members. Cloning and expression of AOX1a , one of the genes encoding alternative oxidase from C. albicans , has previously been reported [Huh and Kang (1999) J. Bacteriol. 181 , 4098–4102]. In the present study we report the isolation of another gene coding for alternative oxidase, designated AOX1b . AOX1b contains a continuous open reading frame that encodes a polypeptide consisting of 365 amino acids. Interestingly, AOX1a and AOX1b were found to be located in tandem on one of the chromosomes of C. albicans . The presence of cyanide in the culture medium remarkably retarded the growth of the aox1a / aox1a mutants. The growth of the aox1b / aox1b mutants and the aox1a / aox1a aox1b / aox1b double mutants was almost completely inhibited in the same medium. β-Galactosidase reporter assays indicated that, whereas AOX1a was expressed constitutively, the expression of AOX1b was dependent on growth phase and was induced by treatment with cyanide, antimycin A, H 2 O 2 , menadione and paraquat. Growth of the cells in media with non-fermentable carbon sources also enhanced the expression of AOX1b . CaSLN1 , which encodes a histidine kinase, seems to be involved in the regulation of AOX expression in C. albicans on the basis of the observation that the activity of cyanide-resistant respiration and the expression level of AOX in the casln1 / casln1 mutants were found to be significantly low under normal conditions and slightly increased in the presence of respiratory inhibitors compared with the wild-type strain. Like AOX1a , AOX1b could also be functionally expressed in AOX-deficient Saccharomyces cerevisiae and confer cyanide-resistant respiration on the organism.