Measurements of tubulin exchange into and from bovine brain microtubules at steady state in vitro were made with 3H-GTP as a marker for tubulin addition to or loss from microtubules. Tubulin has an exchangeable GTP binding site that becomes nonexchangeable in the microtubule. We found that tubulin addition to and loss from microtubules under steady state conditions occurred at equivalent rates, that loss and gain were linear, and that exchange rates (percentage of total tubulin in microtubules lost or gained per hour) were dependent upon microtubule length. Furthermore, we found that podophyllotoxin blocked steady state assembly, but did not alter the rate of steady state tubulin loss. When the assembling microtubule end was pulsed with 3H-GTP at steady state, the label was almost completely retained during a subsequent chase. We conclude that the microtubule assembly-disassembly "equilibrium" is a steady state summation of two different reactions which occur at opposite ends of the microtubule, and that assembly and disassembly occur predominantly and perhaps exclusively at the opposite ends under steady state conditions in vitro.