Exposure of S. cerevisiae cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. This study demonstrates that an additional effect is the triggering of a cell cycle delay during G2/M. Increasing the extracellular osmolarity results in an accumulation of 1N and 2N cells, and a depletion of S phase cells from an asynchronous culture. Additionally, hypertonic stress causes a decrease in mRNA from the B-type cyclin CLB2, phosphorylation of the cyclin dependent kinase Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, while Clb2 protein levels are unaffected. The osmotic stress induced G2 delay is dependent upon the kinase Swe1p. Surprisingly, this delay is not correlated with inhibition of Clb2p-Cdc28p kinase activity. Deletion of SWE1 prevents the phosphorylation of Cdc28p in response to hypertonic shock, and removes the block to cell cycle progression. Deletion of SWE1 also causes synchronized cultures stressed in G2 to accumulate cells with mislocalized nuclei. However, deletion of SWE1 does not prevent the hypertonic stress induced inhibition of Clb2p-Cdc28p kinase activity. Conversely, deletion of HOG1 does prevent Clb2p-Cdc28p inhibition, but does not block Cdc28p phosphorylation or significantly remove the block to cell cycle progression. Deletion of HOG1 does however further disrupt post-stress nuclear localization in combination with a swe1Delta mutation. Finally, preventing Cdc28p phosphorylation by a Y19F substitution does not disrupt the cell cycle delay to the same degree as deletion of SWE1. Together, these results suggest that osmotic stress-induced cell cycle delay is mediated in a novel way by Swe1p, with a contribution from the MAP kinase Hog1p.