Aims To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. Methods and Results The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0.1 mmol l-1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickelnitrilotriacetic acid (Ni2+-NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5.0 and 55 degrees C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5.0 and 70 degrees C. The enzymes had no preferential activities towards a specific cholyl moiety. Conclusions BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. Significance and Impact of the Study This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.