Technical Abstract: Application of nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil around peanut plants has been shown to effectively reduce pre- and post-harvest aflatoxin contamination. Many of our studies conducted over several years demonstrating this effect have utilized inocula consisting of a combination of nontoxigenic A. flavus and A. parasiticus. For purposes of commercializing this process, it would be preferable to produce inocula containing only one strain. Therefore, the purpose of this study was to determine the effect of nontoxigenic strains of A. flavus and A. parasiticus applied alone and in combination on preharvest aflatoxin contamination of peanuts. The study was conducted during two growing seasons in environmental control plots that can ensure optimum conditions for the development of preharvest aflatoxin. Treatments, replicated three times, included an untreated control and formulations of hulled barley coated with conidia of nontoxigenic A. flavus (NRRL 21882), nontoxigenic A. parasiticus (NRRL 21369), and a mixture of the two formulations. Coated barley was applied at a rate of 20 lb/acre 65 days after planting in 2000, and plots were re-inoculated at the same rate 60 days after planting in 2001. All peanuts were harvested, shelled, and analyzed for the presence of toxigenic and nontoxigenic strains of A. flavus and A. parasiticus and for aflatoxins. Results for 2000 showed no significant differences among treatments for total colonization of peanuts by A. flavus and A. parasiticus with colony forming units (CFU)/g ranging from 5.5 × 10 e5 to 8.2 × 10 e5. However, in 2001 untreated peanuts had a significantly higher total colonization (1.2 × 10 e6 CFU/g) than did treated peanuts (average = 1.5 × 10 e5 CFU/g). The incidence of toxigenic strains was significantly less (P < 0.05) in peanuts treated with A. flavus alone in both years (19.9% and 24.3% in 2000 and 2001, respectively) than in controls (69.8% and 95.0%, respectively). The incidence of toxigenic strains was significantly reduced by the mixture in 2000 (18.1%) but not in 2001 (47.8%). Treatment with A. parasiticus alone did not significantly reduce the incidence of toxigenic strains in peanuts (62.8% and 67.2%, respectively). In 2000, aflatoxin contamination was significantly reduced (P < 0.05) by treatment with A. flavus alone (71.3%) and the A. flavus/A. parasiticus mixture (77.1%), but not by A. parasiticus alone. Aflatoxin contamination was significantly reduced (P < 0.05) by all treatments in 2001 with reductions ranging from 89.7% for the A. parasiticus treatment to 92.8% for treatment with A. flavus alone. Regression analysis of data for both years showed a significant (P < 0.01) positive correlation between the incidence of toxigenic strains in peanuts and aflatoxin contamination with an R 2 of 0.70 for 2000 and 0.68 for 2001. As toxigenic strains of A. flavus and A. parasiticus were displaced in peanuts by nontoxigenic strains, aflatoxin levels were reduced correspondingly. Results of this two-year study indicate that application of A. flavus NRRL 21882 to soil was more effective than A. parasiticus NRRL 21369 and equally as effective as a mixture of the two strains in displacing toxigenic strains in peanuts and reducing aflatoxin contamination.