Autolysins are a group of endogenous bacteriolytic enzymes that hydrolyze specific bonds in the cell wall peptidoglycan. The conventional method for determining autolysin activity employs purified autolysinless cell walls as substrate. In this study, preparation of autolysinless whole cells of Bacillus subtilis 168 as an alternative to cell walls is described. The protocol developed is a simple, single step procedure and gives improved yields. The enzyme, autolysin was extracted from B. subtilis 168 using 4 M lithium chloride (LiCl). Substrate was prepared by treating vegetative whole cells of B. subtilis 168 with 10 M LiCl. Autolysin activity was measured as the reduction in optical density of autolysinless whole cells at 600 nm. This method of analysis was applied to estimate the autolysin levels at different growth stages of B. subtilis 168. The autolysin levels were found to be maximum in cells at three stages of growth; start of cell separation, beginning of sporulation and during mother cell lysis. This confirms the established role of autolysins in these cellular processes. (c) 2004 Elsevier Ltd. All rights reserved.