Chemical-based methods have been developed for transformation of DNA into log-phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary-phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log-phase cells but is simpler and more adaptable to high-throughput projects. In this study we have tested different approaches for transformation of early stationary-phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40-fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested. Copyright © 2013 John Wiley & Sons, Ltd.