A novel photoaffinity label, 8-N3-3′-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F1-ATPase from the thermophilic bacterium PS3 (TF1). UV irradiation of TF1 in the presence of 8-N3-3′-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic α and the catalytic β subunits of TF1, demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V1-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the γ subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N3-3′-biotinyl-ATP.