Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl-activated subtilisin-like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin-like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55-60 degrees C, 25-30% NaCl and 70-100 mmol l-1 CaCl2. Enzymes showed stability at 0-30% NaCl and < 20 mmol l-1 CaCl2 and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19-, 34- and 44-kDa proteinases were at 3 center dot 6, 5 center dot 2 and 3 center dot 8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI-TOF and LC-MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F-like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F-like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.