We have developed a high-frequency cotransformation system which is useful in introducing nonreplicating circular DNA plasmids into the fission yeast Schizosaccharomyces pombe. This system depends on two factors: the ability of the ural-complementing helper plasmids pFYM2 and pFYM225 to propagate autonomously in S. pombe, and the intensive recombination activity intrinsic to this yeast. If cotransformed with a helper plasmid, plasmids such as YIp5 or YIp32, Escherichia coli-Saccharomyces cerevisiae shuttle vectors incapable of replication in S. pombe, can enter S. pombe and express the gene carried on them at a frequency comparable to that of autonomously replicating plasmids (10(3) to 10(4) transformants per microgram of DNA). Even if characters of the nonreplicating DNA are not selected directly, 50 to 70% of Ura+ cells transformed with the helper have also incorporated the nonreplicating plasmid. It is shown that these two plasmids have physically recombined at a site of common DNA sequence to form a heteropolymer in the fission yeast. Since any foreign DNA cloned in pBR322 or ColE1 derivatives can be incorporated into S. pombe by using pFYM2 or pFYM225 as a helper, this cotransformation system will serve as a convenient method to examine functional expression of such cloned DNA in S. pombe. This work also demonstrates that the kanamycin resistance gene carried by the bacterial transposon Tn903 can be expressed in S. pombe, as shown by its ability to inactivate the antibiotic G418.