An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.