Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.