The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH3 cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34-41% of tissue culture cell tubulin. Colchicine (10-6 to 10-5 m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.