KcURA3 was cloned from Kluyveromyces cicerisporus CBS4857 by complementation of the ura3 mutation in Saccharomyces cerevisiae. KcURA3 encodes a 267-amino-acid protein with 80% sequence identity to that of S. cerevisiae. An ura3 mutant strain from K. cicerisporus CBS4857, named Y179U, was obtained by selection on 5-fluoroorotic acid plates. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +277. Two vectors, pUK1 and pUKD, bearing KcURA3 were constructed. Either the lithium acetate method or electroporation could be used to transform pUK1 and pUKD into Y179U. The transformation efficiency using electroporation was higher than that using the lithium acetate method.