An improved method for separation of large DNA molecules from Zymomonas mobilis was developed using a contour-clamped, homogeneous electric field technique. The preparation of DNA samples was optimized, as well as the migration conditions, to resolve the chromosomal DNA fractions obtained after digestion with SfiI. A 22 s (45 h) pulse setting gave maximal separation of 240 to 109 kb fragments range, while 9 s (40 h), 3 s (35 h) and 1 s (30 h) settings produced the best separation of 120 to 64 kb, 64 to 34 kb fragments range and less than 35 kb fragments, respectively.