A photoaffinity analogue of taxol, N-([3,5-3H]-4-azidobenzoyl)-N-debenzoyltaxol (7), was synthesized and used to photolabel microtubules. Approximately 20% of the noncovalently bound analogue becomes covalently bound upon irradiation at 300 nm. Incorporated label was stable to a 50% ethanol solution and sodium dodecyl sulfate. About 80% of the incorporated label was found in the β-subunit and 20% in the α-subunit. Incorporation did not occur into unpolymerized tubulin, consistent with the fact that taxol binds only to polymerized tubulin, and was decreased by the presence of taxol. Little or no nonspecific labeling occurs. This analogue is currently being used to identify taxol binding site(s) on tubulin.