Plasmid yield from Acidiphilium strains always had been poor following various standard methods. We adopted some simple modifications in the alkaline lysis procedure to get a better yield of plasmid from these bacteria. An approximately 10- to 20-fold increase in the plasmid yield was achieved when harvested Acidiphilium cells were preincubated 16-20 h at pH 6 in nitrogen-free medium. Another independent approach showed that freezing (-18 to -20 degrees C) of the harvested cells initially and at two subsequent steps in the alkaline lysis procedure of plasmid DNA extraction improved the yield further by 1.5- to 3-fold. The combination of these changes yielded at least 15- to 30-fold more plasmid from various Acidiphilium strains as compared with standard methods.