Mutation of Escherichia coli minicell locus (minB) results in aberrant placement of the division septum. In this paper we report the isolation and characterization of the minB locus. Replacement of the chromosomal minB+ allele by cloned minB sequences containing transposon insertions resulted in the minicell phenotype, indicating that minB+ function is required to maintain the normal division pattern. Paradoxically, overexpression of the locus also resulted in the minicell phenotype. The locus codes for several peptides whose expression is coordinately affected by transposon mutations that also eliminate minB+ function. A subset of the minB peptides is sufficient to prevent minicell formation in minB1 mutants or to induce minicell formation when overproduced in wild-type strains, implicating these peptides in the normal process of localization of the division site. The results indicate that minB is a complex locus whose expression must be maintained within certain limits to maintain the normal pattern of localization of the division septum.