Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.