Glyphosate is often tank-mixed with auxinic herbicide 2,4-D for grass and broadleaf weed control. Here we examined the possible interaction of 2,4-D and glyphosate in barnyard grass, Echinochloa colona (L.) Link. The results showed that 2,4-D antagonizes glyphosate remarkably in glyphosate-resistant populations but only marginally in susceptible populations. This antagonism is related to reduced glyphosate uptake and (to a lesser extent) translocation. As 2,4-D has multiple, unpredictable effects on other herbicides, care must be taken when tank-mixing herbicides with 2,4-D.