Trifluralin is widely used for control of rigid ryegrass in no-till grain crops in southern Australia. Trifluralin must be incorporated into soil to be effective. Several field studies were conducted to evaluate the effect of different tillage systems on vertical seed distribution and efficacy and rate of loss of bioavailable trifluralin. Experiments were conducted during the growing seasons of 2004 and 2005 at two sites in South Australia: on the Roseworthy Campus farm of the University of Adelaide and near Minlaton on the Yorke Peninsula. The tillage systems at the Roseworthy Campus were minimum tillage (MinTill) and no-till using narrow points, whereas four direct-drill-seeding systems were used at Minlaton. Plastic beads were spread on the soil surface to simulate weed seeds. MinTill sowing buried more plastic beads than no-till, indicating that greater soil disturbance was caused by the MinTill system. Trifluralin efficacy was lower under no-till as compared to MinTill. A bioassay, based on response of oat roots, was used to quantify the concentrations of bioavailable trifluralin under different tillage systems. In both years at the Roseworthy Campus, loss of bioavailable trifluralin was greater under no-till than under MinTill; however, the rate of loss under both systems was much faster in 2004 than in 2005. In 2004, bioavailable trifluralin at 12 d after sowing under MinTill and no-till was 55 and 33% of the initial concentration, respectively. In 2005, bioavailable trifluralin remaining at 23 d after sowing was 86% under MinTill and 54% under no-till. At Minlaton in both years, bioavailable trifluralin was greater under tillage systems that provided the highest level of soil disturbance. These results demonstrate that reduced bioavailability of trifluralin occurs in no-till seeding systems, which could result in reduced weed control.