The biological oxidation of ammonia (i.e. nitrification), results in the transformation of relatively immobile NH4+ into a highly mobile NO3-, which is vulnerable to losses through leaching and denitrification, resulting in low nitrogen-use efficiency in agricultural systems. The ability of certain plants to suppress soil nitrifier function by releasing inhibitors from roots is termed 'biological nitrification inhibition' (BNI). Using a recombinant luminescent Nitrosomonas europaea, we developed an assay to detect and quantify the inhibitory effects in plant soil systems termed, 'BNI activity', expressed in ATU (allylthiourea unit). Sorghum roots released (into water-based medium, hereafter referred to as root exudates) substantial amount of BNI activity. The BNI capacity (ATU g-1 root dry wt d-1) of roots changed with growth stage, from 60 ATU at 3 week old to 20 ATU at 45 day stage. In addition, sorghum roots released hydrophobic compounds, which can be recovered by washing with dichloromethane (hereafter referred to as DCM-root wash). One of the active constituents with BNI activity in root exudates, isolated by activity-guided HPLC fractionation, was a flavonoid, identified as sakuranetin. Using a similar approach, the major active constituent with BNI activity in DCM-root wash was isolated and identified as sorgoleone. Both sakuranetin and sorgoleone inhibited Nitrosomonas activity in a dose-response manner. Substantial genetic variability for sorgoleone production capacity was detected in sorghum. Some wild-sorghum species showed high-BNI capacity under field conditions. Potential implications of BNI in inhibiting nitrification and in reducing the nitrogen footprint from agricultural systems will be discussed.