A rapid method used to obtain cell-free extracts from pathogenic mycobacteria was successfully applied to five slowly growing species. The method, which simply consisted of vortexing of the bacterial suspensions with 130-mu-m glass microbeads under predefined conditions, resulted in the breaking of bacterial cells as evidenced by electron microscopy and gave about 1 mg/ml of total proteins. The cell-free extracts so obtained were suitable for SDS-PAGE analysis as well as Western blots. In a pilot test system, this method was applied to Mycobacterium avium (M. av), M. paratuberculosis (P.tb), and related species isolated from "woodpigeon" (M. silvaticum) and Crohn's disease (CD). The procedure was used in the analysis of various mycobacterial proteins by SDS-PAGE and Western blotting, and the findings were compatible with recently published data using criteria based on DNA polymorphism and numerical taxonomy.