Summary: The uptake, translocation and metabolism of glyphosate [N‐(phosphonomethyl) glycine] by Canada thistle (Cirsium arvense (L.) Scop.) (susceptible) and leafy spurge (Euphorbia esula (L.)) (resistant) were examined in an attempt to elucidate the nature of the differential sensitivity. The pattern of uptake and translocation was similar in both species. Glyphosate moved readily in the apoplast and the symplast. High humidity and/or surfactant greatly increased the amount of 14C‐glyphosate absorbed and translocated over that in low humidity and/or without surfactant. No 14Cmetabolites were detected in either species 1 week after treatment with 14C‐glyphosate. More of a glyphosate spray solution containing a fluorescent dye was received and retained on Canada thistle by virtue of its growth habit than on leafy spurge. More glyphosate should therefore be available for uptake by Canada thistle and this may account for the differential sensitivity of these two species.