The HIV protease inhibitor ritonavir (Norvir; ABT-578), currently used in combination with nucleoside analogs and other protease inhibitors in anti-HIV therapy, has previously been quantified by an HPLC procedure. Here, we report the first convenient one-step competitive ELISA for measuring plasma and intracellular ritonavir in HIV patients. Anti-ritonavir antibody was raised in rabbits using ritonavir-KLH conjugate as immunogen, and the enzymatic tracer was prepared by coupling the drug to acetylcholine esterase. Samples for analysis were first extracted with methanol. Bound/free separation was achieved in a microtiter plate previously coated with anti rabbit IgG monoclonal antibody. Fifty percent inhibition was observed at 1 ng/ml ritonavir and the method accurately and specifically detected as little as 3-4 ng/ml of plasma ritonavir as well as intracellular drug in the peripheral blood mononuclear cells of patients undergoing ritonavir therapy. Within-run and day to day coefficients of variation were below 10% and the drugs currently used in HIV therapy did not interfere with the test. The ELISA was applied to the measurement of plasma ritonavir and to the determination of the extracellular/intracellular drug level ratios in HIV patients receiving long-term multidrug therapy.