A protocol was devised to challenge cotton callus with a toxin exuded from sclerotia (SE) of Phymatotrichum omnivorum. Three cultivars (CAMAS, CAMD-E and COKER 310) were grown as callus on a modified Murashige-Skoog medium, and challenged with SE. Resistant calli were increased on nontoxic media, and then reexposed to the SE. Resistant calli were reselected and after the first selection, there was an average of seven resistant microcalli per tube for both CAMAS and CAMD-E. In the second cycle of selection, only a small portion of the inoculated callus pieces died. Resistance of selected calli was evaluated by measuring fresh weight increase on toxic and nontoxic media.