We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin. This gene was predominantly expressed in the interzone II-III region of Vicia faba root nodules. The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression. To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene. Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated. Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II-III region was mediated by the -1252/+10 VfENOD-GRP3 promoter region. This reporter gene expression in V. hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V. faba nodules. An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II-III region was also mediated by the -737/+10 promoter, whereas the -239/+10 promoter only mediated a weak gusAint expression in the interzone II-III region. Since the -239/+10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II-III region of Vicia nodules.