The effects of colchicine on bovine brain microtubules under steady-state conditions have been studied by combined kinetic and equilibrium analysis. Colchicine induces an initially rapid rate of depolymerization when added to microtubules which are at steady state. The initial rate of disassembly follows the kinetics of colchicine binding to free tubulin. However, disassembly is incomplete, and a new steady-state concentration of microtubules is established provided that a sufficient concentration of colchicine-tubulin is present. When steady state is attained from the disassembly direction, colchicine decreases the formation the fraction of tubulin which is participating in the assembly reaction, without measurably changing the apparent critical concentration for polymerization. The extent of depolymerization of microtubules by colchicine is greater the lower the content of microtubule-associated proteins (MAPs). Microtubules at steady state in the presence of either colchicine or GDP do not exhibit subunit flow which occurs in microtubules at steady state in GTP. Colchicine-tubulin will stabilize microtubules in the presence of MAPs but will not support microtubule elongation. Microtubules at steady state in the presence of colchicine depolymerize upon dilution at about the same rate as untreated microtubules, and, in either case, disassembly appears to occur from both ends of the microtubule. These observations appear to be inconsistent with simple reversible assembly mechanisms but may be explained by a model based upon the cooperative interactions of MAP-tubulin oligomers.