AbstractThe simultaneous production of α‐amylase, β‐glucanase and proteolytic enzymes byBacillus subtilishas been studied. The effect of culturing conditions on the simultaneous production of these enzymes has also been investigated. Two known strains were used as standards, the other strains having been isolated and selected in this laboratory for their high enzyme production properties. Of the newly isolated strains three had better enzyme production properties than the standard strains. Selective inhibition was employed to separate total proteolytic activity into two fractions. The strainK160, which was the best strain for enzyme production, produced very few heat stable (100 °C) spores in the culturing conditions used. Enzyme production was correlated with sporulation, which reached its maximum value after about 30 h cultivation. α‐Amylase reached its maximum activity at the same time as sporulation, as did alkaline protease. The maximum for neutral protease activity, however, occurred at an earlier stage, whilst β‐glucanase activity usually continued to increase till the end of the cultivation (48 h). The optimum temperature, agitation and aeration values were determined for the production of enzymes in fermentor cultivation. β‐Glucanase showed remarkably little susceptibility to altered culturing conditions, as did alkaline protease. Sporulation level was always low (5 to 10%) in those cultivations in which enzyme activities reached their