Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.