Glyphosate is a broad-spectrum herbicide that has been used extensively for more than 20 yr. The first glyphosate-resistant weed biotype appeared in 1996; it involved a rigid ryegrass population from Australia that exhibited an LD50 value approximately 10-fold higher than that of sensitive biotypes. We have characterized gene expression levels and glyphosate sensitivity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, in sensitive and resistant lines derived from this population. Restriction fragment length polymorphism analyses were also performed to examine the distribution of EPSPS gene variants and the gene copy number. A two- to threefold increase in basal EPSPS messenger RNA (mRNA) and enzyme activity levels was observed in the most resistant lines analyzed; however, differences among lines in the sensitivity of EPSPS to glyphosate were not apparent. Induction of EPSPS was observed within 48 h after application of 1.5 kg ae ha−1 of glyphosate. This was reflected in elevated levels of both EPSPS mRNA and enzyme activity. Similarly, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mRNA levels increased after glyphosate treatment; however, basal and induced transcript levels were comparable for sensitive and resistant lines in this case. The restriction fragment length polymorphism analyses showed no evidence for gene amplification or cosegregation of a specific EPSPS gene variant with glyphosate resistance. EPSPS expression in lines exhibiting an intermediate level of resistance was indistinguishable from that in glyphosate-sensitive lines, suggesting that the mechanism could, at least in part, be non–target-based.