Binding constants for myristoleic, palmitoleic, palmitic, oleic, and eicosanoic acids and oleyland stearylamine to lipid bilayers have been determined by using microelectrophoresis. Quenching of the fluorescence of the hydrophobic tryptophan analogue N-palmitoyl-L-tryptophan n-hexyl ester incorporated into lipid bilayers by oleic acid and oleylamine and their brominated derivatives is interpreted in terms of unlimited binding to the bilayers. The tryptophan fluorescence of the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum is quenched when reconstituted into bilayers of 1,2-bis(9,10-dibromostearoyl)-phosphatidylcholine (BRPC). Addition of fatty acids, oleylamine, oleyl alcohol, and methyl oleate to the ATPase reconstituted with BRPC reduces the quenching caused by BRPC, indicating binding of these molecules at the lipid-protein interface (annular sites). The charged molecules bind more strongly at the annular sites than do the uncharged molecules. Additional quenching of BRPC-ATPase by brominated derivatives of these molecules indicates binding at sites distinct from the lipid-protein interface, with binding constants similar to those for binding at annular sites, except for oleylamine. Quenching of tryptophan fluorescence of the ATPase by fatty acids and oleylamine suggests that ca. 50% of the tryptophan residues of the ATPase are located close to the lipid-water interface of the membrane.