In situ expression studies show that MsENOD40 is expressed in pericycle cells of alfalfa roots within 3 hr after addition of Sinorhizobium meliloti 1021. This is about 15 hr before cortical cell divisions become apparent. All ENOD40 clones isolated so far share two conserved regions, box 1 and box 2. Translational fusions of GEP to potential open reading frames within the conserved regions of NtENOD40 have been expressed in cowpea protoplasts to study which open reading frames are translated. In this way it was shown that only the first AUG of the transcript, present in box 1, is used as a translational start in cowpea protoplasts. The open reading frame in this conserved region in ENOD40 transcripts encodes a peptide of 10–13 amino acids. In legumes, we suggest a role for the ENOD40 peptide as a signal molecule in the formation of the nodule primordium.