Genistein, as a plant-to-bacteria signal, plays an important role in the establishment of the soybean (Glycine max [L.] Merr.)-Bradyrhizobium japonicum nitrogen-fixing symbiosis. It is essential to the development of effective root nodules and responsible for inducing the nod genes of B. japonicum. Because sub-optimal root zone temperature (RZT) delays infection and early nodule development, and decreases plant nodule number, and genistein addition overcomes some of this, it is reasonable to hypothesize that suboptimal RZT disrupts the inter-organismal signal exchange by inhibiting genistein synthesis. Four experiments were conducted to test these hypotheses. The results of these studies indicated that: (1) when soybean plants were germinated and maintained at RZTs ranging from 13 to 17°C, root genistein concentration and content per plant were lower than those of plants with roots maintained at RZTs above 17°C; (2) when plants were germinated at an optimal RZT (25 °C) then transferred to RZTs below 17°C, and acclimated for a few days, root genistein concentration and content per plant were higher than those of plants with roots maintained either at optimal RZT, or transferred to RZT above 17 °C, although by the end of the experiment, the genistein concentration of root systems at below 17°C RZT appeared to be declining to values below those of plants with above 17 °C RZT; (3) the root genistein concentration increased before the onset of nitrogen fixation and decreased thereafter; and (4) part of the effect of RZTs on genistein content per plant root system was from reductions in genistein concentration at lower RZT5, and part was due to decreased plant root growth.