Glyphosate behavior was examined in Italian ryegrass plants from Chile that were sensitive (S) and resistant (R) to this herbicide. In order to explain the resistance to glyphosate, contact angles, spray retention, foliar uptake, herbicide translocation, and target enzyme activity were studied. Contact angles of glyphosate solutions at a field concentration were 40° to 45° on the abaxial surface of R leaves as compared to 70° on S. Glyphosate spray retention by R plants was 35% lower than by S plants. Glyphosate uptake by the abaxial leaf surface of R plants was about 40% lower than that of S plants. In addition, in the R plants more glyphosate migrated to the tip of the treated leaves. The target enzyme in R and S plants was sensitive to the herbicide. Based on these and previous results, it is concluded that resistance in this Italian ryegrass biotype results from lower spray retention, lower foliar uptake from the abaxial leaf surface, and altered translocation pattern. The decreases in spray retention and foliar uptake constitute new mechanisms of glyphosate resistance.