Protoplasts were isolated from quackgrass to investigate the influence of two surfactants and ammonium sulfate on glyphosate uptake. Without adjuvants, the uptake of glyphosate was not detectable. The uptake of the reference compound 2-aminoisobutyric acid was linear with time and was inhibited 60% in the presence of 10 μM carbonyl cyanide m-chlorophenylhydrazone. Glyphosate uptake increased in the presence of both surfactants and ammonium sulfate. After 0.5 h of incubation the internal glyphosate concentration was 37% of the external concentration (100 μM) in the presence of 0.025 g L−1 Ethomeen T/25, 21% in the presence of 0.25 g L−1 Atplus 201, 13% with 5 mM ammonium sulfate, and 20% with 50 mM ammonium sulfate. The surfactant treatments did not reduce either the viability or the internal pH (= 7.1) as judged by fluorescein diacetate staining or 5,5-dimethyloxazolidine-2,4-dione distribution, respectively. The treatments with ammonium sulfate slightly increased the internal pH. The concentration of Ethomeen T/25 required to disrupt the protoplasts was 0.075 g L−1; Atplus 201 did not disrupt the protoplasts at concentrations up to 10 g L−1.