After nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis, two Bacillus subtilis mutants (div IV-A1 and div IV-B1) were isolated that are defective in the location of division site along cell length. Both mutations were transferred into strain CU403 by transformation, and their properties were studied in the CU403 genetic background. Location of divisions in close proximity to cell pole regions in both mutants results in minicell production. Purified minicells contain a ratio of ribonucleic acid to protein comparable to that found in the parent cells. Autoradiographs of (3)H-thymine incorporation into deoxyribonucleic acid (DNA), thymine-2-(14)C incorporation into DNA, electron micrographs, and chemical analyses for DNA all fail to demonstrate DNA in the minicells. Minicells produced by both mutants are highly motile, an indication of functional energy metabolism. Electron micrographs reveal that minicells are produced by a structurally normal division mechanism and that minicells contain a normal cell surface. The div IV-A1 mutation has been mapped by PBS1 transduction linked to ura. The div IV-B1 mutation is closely linked to pheA by both PBS1 transduction and by co-transformation.