Soluble protein kinase and protein phosphatase activities were localized in the cytosol of pea mesophyll cells using protoplasts fractionation techniques. The molecular weights of the phosphorylated cytosolic proteins, as determined by polyacrylamide gel electrophoresis, were 68, 55, 46, 38, 36, 30, 22 and 12 kDa. Histone and, to a much lesser extent, casein but not phosvitin were accepted as exogenous substrates. In every case serine served as acceptor amino acid for the phosphate residue. The protein phosphorylation activity had an alkaline pH optimum, and showed no response to varying Mg-2, Ca2+, Pi cyclo-AMP or calmodulin concentrations. The kinase activity was competitively inhibited by ADP and pyrophosphate with apparent Ki values of 0.5 and 0.17 mᴍ , respectively. High ATP concentrations (1-4 mᴍ) resulted in a strong decrease of radioactivity in the 32P labeled proteins. It is proposed that the ratio of protein phosphorylation to protein dephosphorylation is regulated by the ATP to ADP ratio in the cytosol.