Biotypes of Lolium rigidum Gaud. (annual ryegrass) resistant to the triazine herbicides were studied to determine the mechanism of resistance. The resistant biotypes have different histories of exposure to the herbicide atrazine but both exhibit greater resistance to the structurally similar triazine herbicide simazine. Simazine resistance is not due to a change at the target site, as a similar concentration of simazine is required for a 50% reduction in electron transport by thylakoids isolated from resistant and susceptible biotypes. Uptake of simazine from nutrient solution and distribution of simazine between the roots and the shoots are similar in resistant and susceptible biotypes. Following application to the roots, more than 95% of the absorbed simazine was translocated to the shoots in both resistant and susceptible biotypes. Resistant biotypes metabolized [ 14C]simazine at a greater rate than susceptible plants when simazine was supplied as either a 12-hr pulse or continuously over 7 days. Over a 7-day exposure to simazine (3 μ M), susceptible plants accumulated simazine in their shoot tissues, whereas resistant plants maintained a low and stable amount of simazine by metabolizing simazine at a greater rate than the susceptible plants. The primary products of simazine metabolism were tentatively identified as N-de-ethyl derivatives. Up to eight other minor metabolites were also observed. The cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) (70 μ M) in combination with simazine (3 μ M) for 7 days caused a greater reduction in dry weight of resistant plants than simazine applied alone. ABT inhibited the metabolism of simazine by all biotypes whether applied as a 12-hr pulse or over a 7-day period. In the presence of ABT the amount of simazine in the resistant shoot tissue was similar to that in susceptible plants treated with simazine alone. The nature of the metabolites and the inhibition of metabolism by ABT suggest the involvement of oxidative enzymes in the mechanism of resistance to simazine.