We previously reported that there were three copies of ATP1 coding for F1-alpha and two copies of ATP3 coding for F1-gamma on the left and right arm of chromosome II, respectively. In this study, we present evidence that there are three closely linked copies of ATP2 encoding the beta subunit of the F1F0-ATPase complex on the right arm of chromosome X in several laboratory strains, including Saccharomyces cerevisiae strain S288C, although it was reported by the yeast genome project that ATP2 is a single-copy gene. Chromosome X fragmentation, long-PCR, chromosome-walking and ATP2-disruption analysis using haploid wild-type strains and prime clone 70645 showed that the three copies of ATP2 are present on the right arm of chromosome X, like those of ATP1 on chromosome II. Each was estimated to be approximately 4 kb apart. We designated the ATP2 proximal to the centromere as ATP2a, the middle one as ATP2b and the distal one as ATP2c. The region containing the three ATP2s is composed of two repeated units of approximately 7 kb; that is, both ends (ATP2a, ATP2c) accompanying the ATP2-neighboring ORFs are the same. A part of YJR119c, YJR120w, YJR122w (CAF17) and YJR123w (RP55), which were reported by the yeast genome project, are contained in the ATP2 repeated units; and the middle ATP2 of the three ATP2s, ATP2b, is located between the two repeated units. Expression of all three copies of ATP2 (ATP2a, ATP2b, ATP2c) was confirmed because a single or double ATP2-disruptant could grow on glycerol, but a triple ATP2-disruptant could not. In addition, of the three copies of ATP1 and ATP2, even if only one copy of the ATP1 and ATP2 genes remained, the cells grew on glycerol.