A chromosomal beta-lactamase gene from Bacillus clausii NR, which is used as a probiotic, was cloned and expressed in Escherichia coli. It encodes a clavulanic acid-susceptible Ambler class A beta-lactamase, BCL-1, with a pI of 5.5 and a molecular mass of ca. 32 kDa. It shares 91% and 62% amino acid identity with the chromosomally encoded PenP penicillinases from B. clausii KSM-K16 and Bacillus licheniformis, respectively. The hydrolytic profile of this beta-lactamase includes penicillins, narrow-spectrum cephalosporins, and cefpirome. This chromosome-encoded enzyme was inducible in B. clausii, and its gene is likely related to upstream-located regulatory genes that share significant identity with those reported to be upstream of the penicillinase gene of B. licheniformis. The bla(BCL-1) gene was located next to the known chromosomal aadD2 gene and the erm34 gene, which encode resistance to aminoglycosides and macrolides, respectively. Similar genes were found in a collection of B. clausii reference strains.