The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic α-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.