The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.