7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.