The fluorescence quenching properties of a series of brominated and iodinated pyrethroids have been used to study the binding of pyrethroids to the (Ca2(+) + Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum. It is suggested that binding at the lipid/protein interface of the ATPase is weak but that binding can occur at other (non-annular sites) on the ATPase. Pyrethroids containing either a brominated fatty acyl or iodinated alcohol moiety quench the tryptophan fluorescence of the ATPase, suggesting that the pyrethroids bound to the ATPase adopt a folded conformation with both the acid and alcohol moieties in contact with hydrophobic regions of the ATPase. Whereas effects of the pyrethroids on the activity of the ATPase in bilayers of dioleoylphosphatidylcholine are small, large increases are observed in the activity of the ATPase reconstituted into bilayers of the short-chain phospholipid, dimyristoleoylphosphatidylcholine (DMPC). The rate of phosphorylation of DMPC-ATPase by ATP is slow, but is increased on addition of pyrethroid. The level of phosphorylation of the ATPase by Pi is reduced on reconstitution into bilayers of DMPC, and this is also increased by addition of pyrethroid.