The role of the elimination of ring B and/or the modification of ring C of colchicine in tubulin binding kinetics and thermodynamics has been characterized, using four different molecules. These ligands are colchicine (COL); 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC), in which the central ring B has been reduced to one bond; allocolchicine (ALLO), in which ring C has been replaced by a six-membered ring; and 2,3,4-trimethoxy-4'-carbsmethoxy-1,1'-biphenyl (TCB), where the same two modifications are made simultaneously. This paper describes the kinetics of association of ALLO with tubulin. The binding is accompanied by a fluorescence increase with slow biphasic kinetics, indicating binding to fast and slow tubulin isotypes. Binding to each of these isotypes occurs in two steps: a fast initial binding followed by a slower isomerization step. The K-1 and k(2) values for ALLO at 25 degrees C are 14 000 +/- 2000 and 25 000 +/- 6000 M(-1) (fast and slow isotypes) and 0.055 +/- 0.003 s(-1) and 0.013 +/- 0.001 s(-1) (fast and slow isotype), respectively. For ALLO the reaction standard enthalpy change of the initial binding is 68 +/- 5 kJ . mol(-1) (fast isotype) and 45 +/- 33 kJ . mol(-1) (slow isotype) and the activation energy for the second forward step is 58 +/- 14 kJ . mol(-1) (fast isotype) and 81 +/- 17 kJ . mol(-1) (slow isotype). Displacement kinetics of bound ALLO by podophyllotoxin was monoexponential. The activation energy for the isomerization in the off direction is 107 +/- 7 kJ . mol(-1). Comparison of the thermodynamic parameters for all four compounds shows that the modifications of both rings are cumulative with respect to overall binding. For the intermediate state there is a mutual influence of both modifications, suggesting an alteration of the reaction pathway.