We investigated the pharmacokinetics and safety of saquinavir/ritonavir when administered with omeprazole simultaneously and 2 h apart to human immunodeficiency virus (HIV) subjects. Saquinavir/ritonavir 12-h pharmacokinetics was assessed with and without omeprazole 40 mg. Subjects were randomized to group A (saquinavir/ritonavir and omeprazole simultaneously/2 h apart) or group B (saquinavir/ritonavir and omeprazole 2 h apart/simultaneously). Saquinavir/ritonavir pharmacokinetics was assessed on days 1, 8, and 22. Within-subject changes were evaluated by geometric mean ratios and 90% confidence interval (CI). Twelve subjects completed the study. GM (90% CI) for saquinavir area under the curve (AUC)(0-12) (ng h/ml), trough concentration (C(trough)) (ng/ml), and maximum concentration (C(max)) (ng/ml) were 14,698 (13,242-20,636), 433 (368-758), 2,513 (2,243-3,329) without omeprazole; 22,646 (18,536-131,861), 750 (619-1,280), 3,890 (3,223-5,133) with omeprazole simultaneously; and 24,549 (20,884-38,894), 851 (720-1,782), 4,141 (3,554-5,992) with omeprazole 2 h earlier. Simultaneous administration of omeprazole significantly increased saquinavir AUC(0-12), C(trough), and C(max) by 54, 73, and 55%, whereas staggered administration by 67, 97, and 65%. No grade 3/4 toxicity or lab abnormalities were observed. In the presence of omeprazole, saquinavir plasma exposure is significantly increased in HIV-infected subjects whether administered simultaneously or 2 h apart.