A DNA extraction suitable for mycobacterial lysis in gentle conditions compatible with genome analysis by pulsed-field gel electrophoresis is presented. Effects of preliminary treatments with SDS, Triton X-100, and hexane on mycobacterial outer layer were observed by electron microscopy. The most efficient procedure, performed on cells from liquid or solid medium, consisted of treatment by Triton X-100, agarose embedding of the cells, and further treatment with alpha-amylase followed by lysozyme and SDS-proteinase K.