The formation of lateral roots (LR) is a major post-embryonic developmental event in plants. In Arabidopsis thaliana, LR development is inhibited by high concentrations of NO3–. Here we present strong evidence that ABA plays an important role in mediating the effects of NO3– on LR formation. Firstly, the inhibitory effect of NO3– is significantly reduced in three ABA insensitive mutants, abi4-1, abi4-2 and abi5-1, but not in abi1-1, abi2-1 and abi3-1. Secondly, inhibition by NO3– is significantly reduced, but not completely abolished, in four ABA synthesis mutants, aba1-1, aba2-3, aba2-4 and aba3-2. These results indicate that there are two regulatory pathways mediating the inhibitory effects of NO3– in A. thaliana roots. One pathway is ABA-dependent and involves ABI4 and ABI5, whereas the second pathway is ABA-independent. In addition, ABA also plays a role in mediating the stimulation of LR elongation by local NO3– applications.