The authors describe an improved method for the high-efficiency transformation of intact yeast cells using single-stranded carrier DNA. This method, previously described, has been further refined to yield 10^6 transformants per microgram of vector DNA, a tenfold improvement, and reduces the time required by 1.5 hours. Key improvements include incubating transformed yeast cells in liquid YPAD for one hour before plating on selective medium, which increased transformation efficiency by about 7-fold. The procedure has also been streamlined by omitting certain steps, such as preincubating cells in TE/LiAc, and scaling down the volumes used. A colony procedure has been developed to further enhance efficiency, yielding up to 1.5×10^6 transformants per μg of vector DNA. The high efficiency of this method has enabled various applications in yeast molecular biology.The authors describe an improved method for the high-efficiency transformation of intact yeast cells using single-stranded carrier DNA. This method, previously described, has been further refined to yield 10^6 transformants per microgram of vector DNA, a tenfold improvement, and reduces the time required by 1.5 hours. Key improvements include incubating transformed yeast cells in liquid YPAD for one hour before plating on selective medium, which increased transformation efficiency by about 7-fold. The procedure has also been streamlined by omitting certain steps, such as preincubating cells in TE/LiAc, and scaling down the volumes used. A colony procedure has been developed to further enhance efficiency, yielding up to 1.5×10^6 transformants per μg of vector DNA. The high efficiency of this method has enabled various applications in yeast molecular biology.