The role of Ca2+ in cold-induced changes in protein phosphorylation, gene expression, and development of freezing tolerance has been studied in cell-suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago sativa spp. falcata cv Anik). Chemical treatments to block Ca2+ channels, antagonize calmodulin action, or inhibit protein kinases markedly inhibited the cellular capacity to develop cold-induced freezing tolerance but had little effect on cell viability. An analysis of phosphoprotein profile by two-dimensional polyacrylamide gel electrophoresis revealed that at low temperature the relative level of phosphorylation of several proteins increased, whereas that of several others decreased. When cold acclimation was carried out in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride, an antagonist of calmodulin and Ca2+-dependent protein kinases, or the Ca2+ channel blocker La3+, the cold-induced changes in protein phosphorylation were strongly inhibited, cells lost their capacity to develop freezing tolerance, and accumulation of transcripts of cold acclimation-specific genes was substantially reduced. An inhibitor of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, had less pronounced effects on the cold-induced protein phosphorylation and caused only a partial inhibition of the cold-induced development of freezing tolerance and accumulation of the transcripts. The level of phosphorylation of one protein, of about 15 kD, increased more than 10-fold at low temperature and showed a strong positive correlation with cold-induced freezing tolerance and gene expression even when the latter were altered with various chemical treatments. These results suggest that Ca2+ and protein phosphorylation, or perhaps a coupling of the two, play an important role during the acquisition of freezing tolerance during cold acclimation.