Colchicine binding and pyrene excimer fluorescence of tubulin have been used to identify cysteine residue(s) essential for the colchicine binding activity of the protein. We report here that both the colchicine binding activity and the ability to form pyrene excimers of tubulin decay at an identical rate when the protein ages at 37 degrees C. Glycerol, which stabilizes the colchicine binding site also stabilizes the excimer formation equally. Thus, these two properties of tubulin are correlated and are likely to belong to the same structural domain. In an attempt to identify the excimer-forming Cys residues, we found that incubation of tubulin with N,N'ethylenebis(iodoacetamide) causes a significant inhibition of excimer fluorescence. Incubation of tubulin with colchicine prior to this treatment fully retains excimer-forming ability. It is known that Cys239 and Cys354 of beta-tubulin, which are about 0.9 nm apart in the native structure, are protected from ethylenebis(iodoacetamide) cross-linking by incubation of tubulin with colchicine [Luduena, R. F. & Roach, M. C. (1981) Pharmacol. Ther. 49, 133-152]. These residues must therefore be responsible for the excimer formation of tubulin with pyrene maleimide. Incubation of tubulin with ethylenebis(iodoacetamide) decreases the colchicine binding activity and the excimer formation at an identical rate. Since the alkylation of Cys239 of beta-tubulin (responsible for tubulin self-assembly) has no effect on colchicine binding [Bai, R., Lin, C. M., Nguyen, N. Y., Liu, T. & Hamel, E. (1989) Biochemistry 28, 5606-5612], our results suggest that excimer formation and the colchicine binding site of tubulin share Cys354 of the beta-subunit. Determination of the number of essential Cys residue(s) for colchicine binding activity, using the statistical method of Tsou [Tsou, C. L. (1962) Sci. Sin. 11, 1535-1558], also shows only one essential Cys residue.