The DNA content of the Bacillus subtilis spore has been redetermined and found to agree with the previous estimate by Fitz-James & Young (1959). This sets an upper limit of 3·0 × 10 9 daltons (1700 μ of the B form) as the amount of DNA in the B. subtilis genome. By autoradiography continuous chromosomal lengths of 800 to 900 μ are shown to occur in vegetative cells. From recent estimates of the size of the genome, these results suggest that it exists as a single chromosome, two of which are present in the spore. However, considering the fragility of the bacterial chromosome, the present results do not rule out the possibility that the B. subtilis genome exists as a much longer chromosomal structure, only one of which is present in the spore.