The 14-3-3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin-dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP-ribosylation of host proteins. In addition, a plant 14-3-3 protein is present in a G-box DNA/protein-binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14-3-3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14-3-3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted. The cDNA encoding a plant 14-3-3 protein under the control of the inducible GAL1 promoter complemented the double disruption. Transfer of the complemented double disruptant to a medium with glucose resulted in the appearance of a high percentage of large budded cells. After prolonged incubation, these cells became enlarged with irregular buds and chains of cells defective in cell-cell separation became visible. These results suggest an essential role of the 14-3-3 proteins, possibly at a later stage of the yeast cell cycle.