PK11195 can activate Pgp-associated ATPase activity and alter Pgp conformation. To show that a nontoxic dose (75 μM) of PK11195 (PK) can specifically stimulate Pgp-associated ATPase activity, we performed assays in which treatment-specific (relative) ATPase activities were calculated (vanadate-sensitive ATPase activities in treated/untreated samples). Assays were performed with microsomal membranes (containing PMs and mitochondria) commercially prepared from Pgp-expressing SF9 insect cells (A), microsomes from Pgp-negative 8226 (gray bars) and Pgp-expressing DOX40 cells (black bars) (B), or PMs from DOX40 cells (C). Like VPL, PK specifically induced ATPase activity in all Pgp-positive models, with dose dependence in DOX40 PMs. Nontoxic doses of CSA (0.1, 1 μM) were ineffective in DOX40 models, but 1 μM CSA induced ATPase activity in Pgp-positive SF9 microsomes and 5 μM CSA induced activity in DOX40 PMs. Data from at least 3 assays are expressed as means plus or minus SEMs. (D) Specific effects of PK11195 on Pgp protein conformation were measured in 3 flow cytometry assays using the Pgp conformation-specific antibody, UIC2 (black bars), and the conformation-insensitive antibody, 15D3 (gray bars). PK11195 increased UIC2 immunoreactivity, but not 15D3 immunoreactivity, in live, Pgp-expressing DOX6, DOX40, KG1a, and VCR cells but not in Pgp-negative 8226 or HL60 parental cells.