Although a well ascertained evidence proves that the activity of the plant plasma membrane H+-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5 ' -AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5 ' -AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H+-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H+-ATPase AHA1 isoform expressed in yeast membranes. Finally, by means of affinity chromatography with immobilized 5 ' -AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5 ' -AMP. The possible role of 5 ' -AMP as a general regulator of 14-3-3 functions in the plant cell is discussed.