We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated K(d) values fell in the range 4-6 microM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.