Spectrofluorometric techniques have been employed to examine the active site of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 126.96.36.199). The fluorescence of both endogenous tryptophan and exogenous 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) has been examined. The fluorescence emission of the enzyme's active site bound MIANS is at 428 nm while the enzyme tryptophan(s) yielded a single fluorescence emission maximum at 347 nm. These values are characteristic of a polar environment for tryptophan and a relatively nonpolar environment for the MIANS. The dynamic fluorescence quenching constants for acrylamide of MIANS and tryptophan are 3.00 M-1 and 1.85 M-1, respectively. Quenching constants for KI of both fluorescent centers were approximately 1 M-1. These data suggest that both fluorophores are poorly accessible to the external anionic contact quencher but that an unchanged quencher, while larger, is still better able to penetrate the enzyme's active site. The extrapolated anisotropies (r0) for ferrochelatase-bound MIANS and tryptophan are 0.198 and 0.307. The dissociation constant (KD) determined by fluorescence anisotropy of protoporphyrin was 1.5 microM with the calculated number of porphyrin binding sites as 1.0 per 40000 daltons. A model is presented for the active site of ferrochelatase based upon the data presented here and previously. This model proposes that the active site is a hydrophobic pocket similar in nature to the heme binding crevices found in many hemoproteins.