1. Cholera toxin has been labelled with the fluorescent probe 4-chloro-7-nitrobenzofuran (Nbf-Cl) in both subunits, and the labelled subunits separated by gel-permeation. They retained their biological activities. 2. Addition of ganglioside GM1 (which binds to subunit B only) to either labelled subunit did not alter the fluorescence of the Nbf probe. 3. Whole toxin was reconstituted using labelled subunit A and unlabelled subunit B. Addition of ganglioside GM1 to the reconstituted toxin enhanced the fluorescence by about 90%, but did not change the wavelength. This enhancement reached a maximum when the ganglioside to toxin ratio was about 1 to 1. Ganglioside GD1b (which does not bind) did not affect the fluorescence. 4. These results suggest that the binding of ganglioside to subunit B alters the environment of the Nbf probe bound to subunit A, presumably by a conformational change.