Protein phosphorylation in vitro was investigated in guard cells from Vicia faba. A number of proteins with apparent molecular masses of 72, 67, 57, 52, 49, 44, 37, and 26 kDa were phosphorylated when guard-cell extract was incubated with [γ- 3 2 P]ATP under Ca 2 + -free conditions. In the presence of Ca 2 + at 1 μM, several proteins with apparent molecular masses of 125, 83, 41, 31, and 25 kDa were newly phosphorylated. These Ca 2 + -dependent protein phosphorylations were suppressed by (8R * , 9S * , 11S * )-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epo xy-1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252a), a wide-range inhibitor of protein kinases, suggesting that the protein phosphorylations were mediated by protein kinases. Several proteins were phosphorylated in vitro in mesophyll extract from Vicia. In contrast to guard cells, there was no detectable Ca 2 + -dependent protein phosphorylation in mesophyll cells. 1-(5-Indonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), an inhibitor of myosin light chain kinase (MLCK), and an antagonist of calmodulin (CaM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited Ca 2 + -dependent phosphorylation of 41- and 25-kDa proteins in guard cells. Fractionation experiments revealed that the Ca 2 + -dependent phosphorylated proteins with molecular masses of 41 and 25 kDa were present in the mitochondria, and the 125- and 31-kDa proteins in the cytosol. These results suggest that Ca 2 + -dependent protein phosphorylation occurs markedly in guard cells, and that Ca 2 + -dependent phosphorylation of 41- and 25-kDa proteins may be catalyzed by MLCK or MLCK-like protein kinase in guard cells.