The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450C-21 in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2 = 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/Kj = Ka = 8.3 +- 0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibition and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.