The embryonically preformed primary root is the first root type of maize that emerges after germination. In this study the abundant soluble proteins of 2.5-day-old primary roots of wild-type and lateral root mutant rum1 seedlings were compared before the initiation of lateral roots. In CBB-stained 2-D gels, among 350 detected proteins 14 were identified as differentially accumulated (>twofold change; t-test: 95% significance) in wild-type versus rum1 primary roots. These proteins which were identified via ESI MS/MS are encoded by 12 different genes. Functionally, these proteins are involved in lignin biosynthesis, defense, and the citrate cycle. Nine of these genes were further analyzed at the RNA expression level. This study represents the first comparative proteomic analysis of maize primary roots prior to lateral root initiation and will contribute to a better understanding of the molecular basis of root development in cereals.