Cysteine synthase (CS; EC 4.2.99.8) activities were compared among crops and grass weeds having different susceptibilities to alachlor. The order of alachlor tolerance of 11 grass species from most to least was: corn ≫ barley=rice=wheat > sorghum=johnsongrass=broadleaf signalgrass > barnyardgrass=large crabgrass > green foxtail=goosegrass, suggesting that corn was tolerant and all other species were susceptible to alachlor. In shoots, rice had the highest extractable CS activity ( 44.3 μmol/min/g fresh weight), and corn, barley, wheat, sorghum, johnsongrass, and broadleaf signalgrass had the second highest activity ranging between 20.6 and 26.4 μmol/min/g fresh weight. CS activity of large crabgrass, green foxtail, and goosegrass was relatively lower (15.5– 18.1 μmol/min/g fresh weight), and barnyardgrass had the lowest CS activity ( 10.9 μmol/min/g fresh weight). Generally, levels of CS activities in roots were similar to those in shoots in each grass species. However, barley had root CS activity lower than that in shoots and green foxtail had root CS activity higher than that in shoots. Alachlor treatment (GR 50 concentrations) had no effect on extractable CS activity in shoots of corn, barley, sorghum, and barnyardgrass. Content of non-protein cysteine in shoots ranged between 60.1 and 131.5 nmol/g fresh weight, and was not largely different among the grass species except for green foxtail where it was high, i.e., 334.6 nmol/g fresh weight. Content of other non-protein thiols was highest in corn (574.7 nmol/g fresh weight), which was tolerant to alachlor, and the contents were relatively low in other grass species which were susceptible. Alachlor susceptibility was more highly correlated to CS activity ( R 2=0.514) in shoots than to the contents of cysteine ( R 2=0.309) and to other thiols ( R 2=0.025) in grass species susceptible to alachlor. In addition, CS activity in roots ( R 2=0.159) was less correlated to the selectivity than shoots. These results indicate that CS activity in shoots could contribute to alachlor selectivity in grass species.