The authors have previously shown that cold acclimation and cold acclimation-specific (cas) gene expression in alfalfa require cold-triggered calcium influx and phosphorylation of specific pre-existing proteins. In this study, the authors used the expression of cas15 gene as an end-point marker to examine the role of protein phosphorylation in low temperature signal transduction in alfalfa cells. Whereas the protein kinase inhibitor staurosporine prevented the cold induction of cas15, the protein phosphatase inhibitor okadaic acid induced the cas15 at 25°C. Upon exposure of cells to cold, total cellular protein phosphatase activity rapidly declined by 30% but all of this decline could be attributed to an almost complete inhibition of protein phosphatase 2A (PP2A). This cold-inactivation of PP2A was found to be mediated by calcium influx and could be reproduced at 25°C by treating the cells with the calcium ionophore A23187 or with the calcium channel agonist Bay K8644. On exposure of cells to cold, the transcript and protein levels of the PP2A catalytic subunit (PP2Ac) did not decline, but the binding of anti-PP2Ac antibody to native PP2Ac increased, indicating a demasking of the PP2Ac epitope. The possible mechanisms by which cold might inhibit PP2A are discussed.