An improved method for high efficiency transformation of intact yeast cells is described. The method involves using single-stranded carrier DNA and has been optimized to achieve a transformation efficiency of 10^6 transformants per microgram of vector DNA, a tenfold improvement over previous methods, and reduces the time required by 1.5 hours. The method has been scaled down to various levels, and a colony procedure has been developed. Incubating transformed yeast cells in liquid YPAD for one hour prior to plating on selective medium increased the transformation efficiency about 7-fold. It was found that preincubation in TE/LiAc was not necessary, and PEG/TE/LiAc could be added immediately after cells were aliquoted into tubes containing vector and carrier DNA. This shortened procedure resulted in a transformation frequency of up to 1.2 × 10^6 colonies per μg of plasmid DNA. The volumes have also been scaled down compared to the original protocol. The colony procedure involves scraping off a large colony or several medium-sized colonies from a YPAD plate, washing them in sterile water, resuspending them in 1 × LiAc/TE, and transforming them as in steps 5 to 11 in Figure 1. This procedure gives up to 1.5 × 10^4 transformants per μg of vector DNA. The high efficiency of the previously developed transformation method made certain applications to some of the most important needs of current yeast molecular biology possible. The work was supported by grants from various organizations. The method is detailed in a protocol involving several steps, including inoculating cells into YPAD medium, diluting and regrowing cells, washing and resuspending cells in TE/LiAc, mixing with transforming DNA and carrier DNA, adding PEG/TE/LiAc, incubating, heat shocking, and plating on selective medium.An improved method for high efficiency transformation of intact yeast cells is described. The method involves using single-stranded carrier DNA and has been optimized to achieve a transformation efficiency of 10^6 transformants per microgram of vector DNA, a tenfold improvement over previous methods, and reduces the time required by 1.5 hours. The method has been scaled down to various levels, and a colony procedure has been developed. Incubating transformed yeast cells in liquid YPAD for one hour prior to plating on selective medium increased the transformation efficiency about 7-fold. It was found that preincubation in TE/LiAc was not necessary, and PEG/TE/LiAc could be added immediately after cells were aliquoted into tubes containing vector and carrier DNA. This shortened procedure resulted in a transformation frequency of up to 1.2 × 10^6 colonies per μg of plasmid DNA. The volumes have also been scaled down compared to the original protocol. The colony procedure involves scraping off a large colony or several medium-sized colonies from a YPAD plate, washing them in sterile water, resuspending them in 1 × LiAc/TE, and transforming them as in steps 5 to 11 in Figure 1. This procedure gives up to 1.5 × 10^4 transformants per μg of vector DNA. The high efficiency of the previously developed transformation method made certain applications to some of the most important needs of current yeast molecular biology possible. The work was supported by grants from various organizations. The method is detailed in a protocol involving several steps, including inoculating cells into YPAD medium, diluting and regrowing cells, washing and resuspending cells in TE/LiAc, mixing with transforming DNA and carrier DNA, adding PEG/TE/LiAc, incubating, heat shocking, and plating on selective medium.