Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 A degrees C but not at 42 A degrees C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.