Using the one-step gene disruption technique, we studied the effect of various parameters on the disruption frequency (percentage of homologous integrants) and transformation efficiency (number of transformants per microgram of input DNA) of integrative transformation in Schizosaccharomyces pombe. We used suc1 as the target gene for disruption and ura4 as the selectable marker. Our results are as follows. 1) Use of the strong adh1 promoter to drive the expression of ura4 did not affect the disruption frequency but modestly increased the transformation efficiency. 2) The transformation method had a profound effect, with the lithium acetate method yielding both a 10-fold higher disruption frequency compared to the protoplast method and a 5- to 10-fold higher transformation efficiency. 3) The presence of increasing amounts of non-homologous sequences at the ends of the transforming DNA decreased the disruption frequency by up to 5-fold but had no effect on the transformation efficiency. We also describe the use of the sup3-5 allele in an ade6-704 genetic background to discriminate between the products of homologous versus non-homologous integration, thereby promoting the identification of rare homologous integrants.