Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.