The cause of differential susceptibility of barnyardgrass, hemp sesbania, pitted morningglory, and prickly sida to glyphosate was examined by measuring the absorption of 14C-glyphosate, quantifying the amount of epicuticular wax, and observing the wettability of leaf surfaces. In greenhouse experiments, the biomass of barnyardgrass and prickly sida was reduced by 95% by Roundup Ultra®. Hemp sesbania and pitted morningglory showed more tolerance, with 66 and 51% average biomass reduction, respectively. Absorption of 14C-glyphosate in a controlled environment did not follow the trend in species susceptibility with barnyardgrass, 30%; prickly sida, 18%; hemp sesbania, 52%; and pitted morningglory, 6%; absorption. The high tolerance of pitted morningglory to glyphosate can be attributed mostly to limited absorption, but the tolerance of hemp sesbania is due to other mechanisms. The addition of nonionic surfactant (NIS) to a low rate of Roundup Ultra® reduced absorption of 14C-glyphosate by barnyardgrass and hemp sesbania, but had no effect on the herbicidal activity. Glyphosate absorption in the four weed species was not correlated with quantity of chloroform-extracted wax or leaf wettability. Pitted morningglory and prickly sida, which contained the least leaf wax, also had smaller contact angles or higher leaf wettability than the species with more waxy leaves. The adjuvant in Roundup Ultra® reduced contact angles of the four species compared to contact angles obtained using deionized water alone. The addition of 0.25% v/v NIS alone to water reduced contact angles more than did the adjuvant in Roundup Ultra® solution.