Several isoflavonoid phytoalexins produced by soybeans are known to be estrogenic, with potential beneficial health effects in humans. Increased production of phytoalexins by the soybean plant will facilitate research efforts in this area. In this study, phytoalexin induction and accumulation in soybean cotyledon tissue was observed using four species of Aspergillus: A. sojae, A. oryzae, A. niger, and A. flavus. All four Aspergillus species tested elicited phytoalexin accumulation in living soybean cotyledons. Results from a time course study indicated that maximum concentrations of the phytoalexin glyceollin, 955 microg/g fresh weight (fw), occurred at day 3 in soybean cotyledon tissue inoculated with A. sojae. Other Aspergillus species caused an accumulation of glyceollin at significantly lower levels. A maximum concentration of coumestrol of 27.2 microg/g fw was obtained from soybean cotyledons inoculated with A. niger. Soybean phytoalexins induced by food-grade A. sojae and A. oryzae allowed the collection of higher concentrations of phytoalexins for further examination in several in vitro and in vivo biological studies conducted to determine potential estrogenic activities.