A method is described for the purification of 3,3-dihydroxyazetidine (DHA), which accelerates the growth of Bifidobacterium spp., from the culture medium of Bacillus mesentericus (BM). Purification involved adsorption to an ion-exchange resin; it required less time and gave a higher yield than a previously reported method. Monitoring the inhibition of E. coli NIHJ JC-2, we searched for other strains that produced 3,3-dihydroxyazetidine. We found that not only B. mesentericus TO-A but also B. subtilis IFO13719 produced the compound of interest.