Chloroplast F1-ATPase (CF1) was photolabeled by a radiolabeled photoactivatable derivative of Pi, 4-azido-2-nitrophenyl [32P]phosphate (ANPP). The radioactivity was localized in the beta subunit of CF1. Upon cleavage of the beta subunit by cyanogen bromide, the predominantly labeled peptide was recovered, which was subsequently subjected to tryptic digestion. A tryptic peptide (spanning Ile312-Arg354), was found to contain nearly all the covalently bound radioactivity. By Edman degradation, the labeled amino acid residues were identified as Tyr328, Val329 and Pro330. The labeled beta-Tyr328 of CF1 is the equivalent of beta-Tyr311 of F1 from beef heart mitochondria, which was previously found to be photolabeled by ANPP [J. Garin et al. (1989) Biochemistry 28, 1442-1448].