Calcium-dependent protein kinases (CPKs) are major plant Ca2+ sensors, many of which have roles in plant stress responses. The Arabidopsis genome encodes 34 CPK isoforms. Here we report characterization of AtCPK32 gene function. Analysis of transgenic plants expressing pCPK32-GUS shows that CPK32 is highly expressed in roots, pollen and embryo, as well as leaf hydathodes, and the abscission zone of mature siliques. Real time RT-PCR and promoter expression patterns show that CPK32 is responsive to abiotic and biotic stresses. Plants treated with salt, ABA, osmotic stress (PEG), wounding, and flagellin 22 peptide show up-regulation of CPK32 upon these stress treatments. The overexpression of CPK32 results in ABA and salt insensitive phenotypes whereas disruption of CPK32 gene by T&ndashDNA insertion leads to ABA and salt hypersensitive phenotypes in seed germination and early seedling growth assays. Interestingly, CPK32 overexpression plants are sensitive to drought whereas cpk32&ndash1 mutant plants are drought resistant suggesting that ABA and salt might be operating independent of drought stress tolerance. In a protoplast transient expression assay CPK32 is localized to the plasma membrane. Upon ABA treatment, CPK32 quickly moves from the plasma membrane to the cytosol and nucleus. Two key posttranslational modifications, myristoylation and palmitoylation play a crucial role in sub-cellular targeting of CPK32 to the plasma membrane. Mutation of these acylation sites leads to cytosolic and nuclear localization of CPK32 protein. Together our data provides evidence that CPK32 is a negative regulator of ABA signaling and is involved in multiple stress signaling pathways.