During the early 1980s, Bauer and associates reported that nodulation potential in primary roots of soybean seedlings following inoculation with rhizobia was significantly reduced in developmentally younger regions. They suggested that this phenomenon might be due to a fast-acting regulatory mechanism in the host that prevented excessive nodulation. However, the molecular mechanism of this fast-acting regulatory response remains uncertain. Here, we sought to elucidate components of this regulatory mechanism by investigating the expression of the NSP1 and NSP2 genes that encode a GRAS transcription factor required for nodule initiation. First, we confirmed that younger regions of Lotus japonicus roots also show a reduction in nodule numbers in response to Mesorhizobium loti. Then, we compared the expression levels of NSP1 and NSP2 in developmentally younger regions of primary roots. After inoculation with M. loti, expression of NSP1 was transiently induced whereas that of NSP2 was significantly down-regulated 1 d after inoculation. This result implicates that down-regulation of NSP2 might cause a fast-acting regulatory mechanism to prevent further nodulation. Next we overexpressed NSP2 in wild-type plants. Overexpression resulted in the clustering of nodules in the upper region of the root but strong suppression of nodulation in the lower region. In contrast, overexpression of NSP2 in har1 hypernodulating mutants resulted in an increased number of nodule primordia even in the root tip region. These results indicate that HAR1 negatively regulates NSP2-induced excessive nodule formation in the developmentally younger regions of roots.