To understand the mechanisms of toxicity of 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), we have studied their effects on the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), DNA synthesis, and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. Exposure of cells to 2,4‐D or 2,4,5‐T resulted in a dose‐dependent inhibition of DNA synthesis; 50% inhibition occurred at 2.21 mM and 0.90 mM for 2,4‐D and 2,4,5‐T, respectively. Furthermore, a strong synergistic inhibition of DNA synthesis was produced by mixtures (each having a total concentration of 1.25 mM) of 2,4‐D with 2,4,5‐T. Similarly, 2,4,5‐T is more potent than 2,4‐D in causing cytoskeletal perturbation as revealed by fluorescence microscopy. Treatment of cells with 2,4‐D (2.5 mM) or 2,4,5‐T (1.25 mM) for 20 h resulted in severe MT aggregation and the appearance of large bundles, which were organized in a rope‐like structure in the former and a dramatic octopus‐like pattern in the latter. Further, MT bundling is particularly severe in the cell center. Under these conditions, marked changes in MF organization also occurred as evidenced by clustering and crisscrossing of MF in the perinuclear region. A 1:1 mixture (final = 1.25 mM) of 2,4‐D and 2,4,5‐T, a formulation equivalent to Agent Orange composition, also induced a dramatic perturbation to the organization of MT and MF, resulting in the formation of ring‐like structures. MT bundling is still apparent, especially around the outer edge of the “rings.” MF are localized predominantly along the cell periphery, where they appear to be aggregated tightly forming patches. Surprisingly, the synthesis and composition of cytoskeletal proteins, which are resistant to detergent extraction but released by CaCl2, are essentially unaffected by 2,4‐D or 2,4,5‐T. These results suggest that the dramatic perturbation of the cytoskeletal morphology caused by these herbicides probably only results from a structural reorganization and redistribution of MT and MF.