Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N'-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with N,N'-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N'-dicyclohexylcarbodiimide, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis[3-propyl-5-oxoisoxazol-4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H(+)-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.