Abstract: Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21 pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1 ppz2 double disruptant showed temperature-sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl(2). The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl(2). The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases.CI - Copyright 2002 John Wiley & Sons, Ltd.