We have examined the intrinsic fluorescence properties of a highly purified chloroplast H(+)-ATPase (CF0F1) preparation [R. D. Kirch and P. Graber (1992) Acta Physiol. Scand. 746, 9-12). Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluorescence both at 277-nm excitation, favoring tyrosine excitation, and at 295-nm excitation, favoring tryptophan excitation. A broad tryptophan fluorescence peak was observed with a maximum at around 335 nm and a broad shoulder around 350 nm. Denaturation of the enzyme complex with guanidine-HCl resulted in a significant increase (approximately 40%) in tyrosine fluorescence. The fluorescence spectrum (lambda ex = 295 nm) of the inhibitory epsilon subunit isolated from CF1 resembled that of CF1, indicating the presence of two tryptophan species located in different environments. Fluorescence quenching by potassium iodide indicated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of epsilon from CF1. Thus, when epsilon binds to CF1, a tryptophan residue becomes partially buried, probably at an interface between epsilon and another (possibly gamma) CF1 subunit. Removal of the epsilon subunit from CF1 leads to an increase in tyrosine fluorescence of a magnitude similar to that obtained upon denaturation of the CF0F1 complex. The results suggest that the reversible association of the epsilon subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescence of the enzyme complex.