Microtubule assembly in diploid human skin fibroblasts was studied by [3H]- colchicine binding to disaggregated microtubule subunits and to total cell tubulin. Microtubule content per milligram of cell protein was critically dependent upon cell density. As cultures neared confluence, microtubules increased and total cell tubulin decreased ; the percent of tubulin assembled into microtubules increased from 5 .3 in sparse cultures to 58.3 in confluent cultures. Microtubules disappeared with a half-time of 2 min in response to 0°C incubation and reformed upon rewarming. Brief treatment of intact cells with concanavalin A or cytochalasin A depolymerized microtubules to 55 or 56% of control levels. The effect of concanavalin A was prevented by a-methylmannoside. Fibroblast microtubule assembly was not significantly altered by cyclic nucleotides, ascorbate, glucose, insulin, medium calcium concentration, or calcium ionophore A23187.