In order to label a noncatalytic site, F1 containing one vacant noncatalytic site was incubated with a large molar excess of 2-azido-(US ,el-TSP)ATP. Unbound ligand was removed on a Sephadex centrifuge column and MgATP added to displace 2-azido-ANP from catalytic sites. Unbound ligand was again removed on a centrifuge column. Alternatively, F1 was first incubated with MgATP to fill all noncatalytic sites, passed through a centrifuge column, and then incubated with a stoichiometric amount of the 2-azido analog. This resulted in the specific loading of analog at a single high-affinity catalytic site under conditions for uni-site catalysis. The complexes were photolyzed and in both cases, the US subunit was modified. However, separation of tryptic peptides by reversed phase HPLC revealed different labeled peptides. The major labeled peptides were isolated and sequenced. Photolabeling of the noncatalytic site resulted in the modification of Tyr 368, whereas the major labeled tryptic peptide obtained for the catalytic site contained Tyr 345. Near complete inhibition of ATPase activity required the covalent incorporation of either one mol of the photoaffinity analog at a catalytic site or two mol at noncatalytic sites per F1.