Extracellular signals produced by Rhizobium meliloti are able to induce root hair deformations and nodule organogenesis on alfalfa. The production of these signals is controlled by bacterial nod genes. To enable their isolation in significant amounts, an overproducting strain was constructed. These Nod factors were first extracted by butanol from the culture medium and further purified by reverse-phase high performance liquid chromatography, ion-exchange, and Sephadex LH-20 chromatographies. The structure of the major signal, called NodRm-1, was determined by mass spectrometry, nuclear magnetic resonance, 35S labeling, chemical analysis, and enzymatic degradation, and was shown to be a sulfated and acylated tetramer of glucosamine namely, beta-D-GlcpN(2,9-hexadecadie-noyl) - (1----4) - beta - D - Glc p Nac - (1----4) - beta - D - Glc p NAc - (1----4) - D - GlcpNAc-6-SO3H. Another Nod factor (called Ac-NodRm-1) was co-purified and identified as NodRm-1 acetylated on the C-6 of the nonreducing end sugar. NodRm-1 elicits root hair deformation specifically on alfalfa at a concentration less than 10(-10) M but has no effect on vetch (a heterologous host plant).