one platethreereporter assay format for the detection and validation of yeast twohybrid interactions we describe a novel assay format for the gal4based yeast twohybridsystem in which the readout from three different reporter genes is measured sequentially in a single microplate activation of the ura3 mel1 and lacz reporters in response to a proteinprotein interaction is monitored by measuring sequentially i growth in medium lacking uracil ii alphagalactosidase activity and iii betagalactosidase the data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activationstrength that may be confirmed by further analysis the assay procedure is inexpensive and requires few liquidhandling steps it is appropriate for automated highthroughput interaction mating assays validation of putative interactor strains and hybridprotein selfactivator tests