Field bindweed biotypes varying in susceptibility to glyphosate were studied to determine the role of glyphosate absorption and translocation in susceptibility differences. Absorption and translocation of radiolabeled glyphosate were measured after application to leaves of glyphosate-tolerant and -sensitive field bindweed biotypes grown under ambient, low, or high relative humidity (RH). Glyphosate injury and cuticle mass were also determined at both low and high RH. To avoid potential differences in susceptibility due to differential foliar absorption, glyphosate was fed to roots of hydroponically grown plants, and uptake, translocation, and injury patterns were also determined. Under ambient RH, no differences in foliar-applied glyphosate absorption or translocation were detected between field bindweed biotypes. The RH under which plants were grown influenced cuticle amounts, glyphosate absorption, and injury. Plants of both biotypes had less cuticular material when grown at high RH (80%) than at low RH (30%); however, more radioactivity was absorbed by the susceptible biotype at low RH. Regardless of RH regime, glyphosate injury was greater in the susceptible biotype than in the tolerant biotype. Glyphosate fed to roots of hydroponically grown plants was absorbed and translocated equally in the biotypes, yet relative differential glyphosate sensitivities were retained. These results suggest that differences in glyphosate susceptibility between field bindweed biotypes cannot be fully accounted for by differences in glyphosate absorption or translocation.