We have identified two single-copy genes from the model legume. Medicago truncatula (MtENOD16 and 20) whose expression can be correlated with early stages of root nodulation and whose predicted coding sequences are partially homologous to both pea/vetch ENOD5 and soybean N315/ENOD55. Database searching and sequence alignment have defined the encoded early nodulins as a distinct sub-family of phytocyanin-related proteins, although the absence of key ligands implies that they are unlikely to bind copper. Molecular modelling based on known phytocyanin structure has been used to predict the 3-dimensional conformation of the principle globular domain of MtENOD16/20. Additional structural features common to both early nodulin and phytocyanin precursors include an N-terminal transit peptide, a highly variable (hydroxy)proline-rich sequence which probably undergoes extensive post-translational modification, and a hydrophobic C-terminal tail.