When logarithmically growing Bacillus subtilis cells were exposed to the antitumor protein, neocarzinostatin (NCS), at a concentration of 50 mug/ml, cellular DNA was gradually degraded into an acid-soluble form (up to 60 percent of total DNA). The degradation appeared to initiate at the growing regions of DNA and to proceed sequentially from the nascent regions to preexistent DNA. Concomitantly with, or perhaps as a consequence of, the degradation of growing regions, DNA detached from the cell membrane and started to show single-strand nicks within 30 minutes after exposure of the cells to NCS, whereas double strand scission in the DNA became detectable in about 90 minutes. Such endonucleolytic breaks in DNA eventually gave rise to the formation of double-stranded DNA fragments of a single-size class (30-S) as determined by sedimentation in either neutral or alkaline sucrose gradients. In contrast to previous results with Sarcina lutea, the NCS-induced DNA degradation was stimulated by chloramphenicol in B. subtilis and the DNA fragment were not the final breakdown products, but were further degraded into acid-soluble materials.