Seventy isolates of Verticillium dahliae, five isolates of Verticillium albo-atrum, and one isolate each of Verticillium nigrescens, Verticillium nubilum, and Verticillium tricorpus were compared for virulence to cotton and sensitivity to the alkaloid sanguinarine. Virulence of the V. albo-atrum and V. dahliae isolates to cotton was correlated with their growth rates on media containing sanguinarine. In the presence of sanguinarine 24 defoliating isolates all developed a blue fluorescence, which indicates detoxification to dihydrosanguinarine. Only 10 of 51 mild to moderately virulent strains fluoresced blue under the same conditions. Initial rates of uptake of sanguinarine and its toxicity to conidia was greater at pH 6.5 than at 4.5. After 18 h at pH 4.5, isolates tolerant to the alkaloid had removed 4–5 times as much sanguinarine from the medium as sensitive isolates; at pH 6.5 the amount of sanguinarine uptake was similar for all isolates. Uptake of sanguinarine from the medium occurred concurrently with an increasing blue fluorescence of intracellular areas in sanguinarine-tolerant isolates. These tolerant isolates of V. albo-atrum and V. dahliae appear to detoxify sanguinarine and accumulate the blue-fluorescent dihydrosanguinarine. A similar system may occur in the cotton – V. dahliae host–parasite relationship.