The exact molecular mechanism of inhalational anesthetics remains obscure. Since the enzyme activity of the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle fibres is modified by halothane and because protein-protein interactions play an important role in the regulation of Ca2+-regulatory proteins, we investigated the effect of this volatile drug on the oligomerization of the fast-twitch Ca2+-ATPase. Using electrophoretic separation following incubation with halothane, increases in relative molecular mass were determined by immunoblotting with a monoclonal antibody to the SERCA1 isoform of the Ca2+-ATPase. Distinct drug-induced decreases in electrophoretic mobility indicated oligomerization of the native Ca2+-pump by halothane, comparable to crosslinking-mediated formation of homo-tetramers. Determination of the effect of halothane on enzyme activity suggested that halothane-mediated protein aggregation triggers a partial inhibition of Ca2+-pump units. Thus, halothane appears to exert its action via specific peptide binding sites and not indirectly by lipid perturbation These findings support the protein theory of anesthetic action.