Maximum velocity and Km(PEP.Mg) of phosphoenolpyruvate carboxylase (PEPC) from stomatal guard cells of Vicia faba L. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparent in vivo activity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, post-translational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 x 10(-9) g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata. Vmax at pH 7.0 was not significantly different from that at pH 8.5. Neither Vmax nor Km(PEP.Mg) was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However, Km(PEP.Mg) was greater than 4x lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was greater than 3x greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.