The activity of an auxin-stimulated NADH oxidase activity from soybean hypocotyls was inhibited by submicromolar concentrations of ATP. Auxins are plant growth regulators that increase the rate of cell enlargement in plant stems. A synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was used. The inhibition was half maximal at 1 nM ATP and was not observed with other nucleotides and nucleosides. The inhibition was the result of an increase in the Km for NADH from about 60 μM to > 100 μM and was noncompetitive. The decrease in Km due to ATP was enhanced by the addition of 1 μM 2,4-D. The Vmax of the plasma membrane NADH oxidase was approximately doubled (1.5–2.8-fold) by ATP and by 1 µM 2,4-D. No further increase in the Vmax was observed by the combination of 1 nM to 0.1 mM ATP in the presence of 1 μM 2,4-D. The results demonstrate a response of the NADH oxidase activity of isolated vesicles of soybean plasma membranes to ATP distinct from that observed previously with other nucleotide di- and triphosphates. The results are suggestive either of control of the cell surface NADH oxidase by phosphorylation or a direct response to ATP binding at nanomolar concentrations of ATP.