Expression of bovine PKCα in Saccharomyces cerevisiae results in growth inhibition, which is strongly augmented upon addition of phorbol esters. To investigate the nature of this PKC-induced inhibition of cell growth, wildtype and bovine PKCα-expressing yeast cells were examined by flow cytometry and by fluorescence microscopy after staining with propidium iodide. Upon expression and activation of the mammalian PKC isoform, cells accumulated in the G2/M phase of the cell cycle and exhibited impaired chromsome segregation. In some instances, PKC expression and activation was accompanied by a defect in septum formation between mother and daughter cells. cDNA microarray analysis revealed 4 genes (CTS1, DSE1, DSE2, and SVS1) that changed expression in both a PKCα- and phorbol ester-dependent manner. These findings were confirmed by quantitative real-time PCR. Three of these genes are involved in cell wall turnover and are regulated by a single transcription factor (Ace 2) that localizes to daughter cell nuclei after cytokinesis. Taken together, these observations suggest that expression and activation of bovine PKCα in yeast cells repress growth by inducing an accumulation of cells in G2/M, likely through an impairment of chromosome segregation, cytokinesis, and septum formation. Moreover, when these observations are taken in the context of previously published observations with various yeast null mutants, we propose that bovine PKCα may directly or indirectly activate a subunit of the PP2A phosphatase complex (cdc55), which is a component of the mitotic spindle checkpoint.