Plant resistance to glyphosate has been reported far less frequently than resistance to sulfonylurea and imidazolinone herbicides. However, these studies tend to be anecdotal, without side by side comparisons for a single species or natural isolate. In this study, we tested the frequencies of resistance of three herbicides in a controlled ethylmethanesulfonate (EMS) saturation mutagenesis experiment, allowing a direct comparison of the frequencies at which resistant mutant plants arise. The 100% growth inhibition dose rates of glyphosate, chlorsulfuron (a sulfonylurea herbicide), and imazethapyr (an imidazolinone herbicide) were determined for Arabidopsis. Populations of EMS-mutagenized M 2 seedlings were sprayed with twice the 100% growth inhibition dose of glyphosate, chlorsulfuron, or imazethapyr, and herbicide-resistant mutants were identified. Although there were no glyphosate-resistant mutants among M 2 progeny of 125,000 Columbia and 125,000 Landsberg erecta M 1 lines, chlorsulfuron resistance and imazethapyr resistance each appeared at frequencies of 3.2 ϫ 10 Ϫ5 . Given the observed frequency of herbicide resistance mutations, we calculate that there are at least 700 mutations in each EMS-mutagenized Arabidopsis line and that fewer than 50,000 M 1 lines are needed to have a 95% chance of finding a mutation in any given G:C base pair in the genome. As part of this study, two previously unreported Arabidopsis mutations conferring resistance to imidazolinone herbicides, csr1-5 (Ala-122-Thr) and csr1-6 (Ala-205-Val), were discovered. Neither of these mutations caused enhanced resistance to chlorsulfuron in Arabidopsis.Spontaneous herbicide resistance is generally thought to occur within weed populations as a consequence of the intense selective pressure exerted by a lack of diversity in weed management practices (Gressel and Segel, 1978). Factors such as extended residual soil activity, lack of rotation to other herbicidal modes of action, and specific managerial practices further discriminate between resistant and susceptible individuals within a population (Powles and Holtum, 1994). In addition, the rate and severity at which resistant weed infestations occur is influenced by genetic and ecophysiological determinants such as the mode of inheritance of a given resistance mechanism, the absence or presence of fitness penalties associated with resistance, and the reproductive habit of a given weed species (Gressel and Segel, 1978;Jasieniuk et al., 1996; Gardner et al., 1998). To date, more than 261 herbicide-resistant weed biotypes exist distributed among 52 different countries, involving at least 17 different herbicide modes of action (Heap, 2002). Because application rate and other factors vary greatly in the field, it is difficult to make a direct comparison of the frequencies at which weeds develop resistance to different herbicides. To circumvent this problem, we have used a controlled laboratory setting to compare the frequencies at which heavily mutagenized populations of Arabidopsis develop resistanc...