Vascular immunotargeting to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) facilitates drug delivery to endothelium. We used human PECAM-transfected REN cells (REN/PECAM) as a model to compare targeting of antioxidant enzyme catalase conjugated with PECAM antibody (anti-PECAM/catalase) with adenoviral catalase delivery. Anti-PECAM/(125)I-catalase bound to REN/PECAM, but not to REN cells (70 vs. 1 ng/well vs. < 2 ng/well of unmodified catalase). At a virus-to-cell ratio of 1, elevated levels of catalase protein were detected by immunoblotting after adenoviral transfection of REN/PECAM and REN cells alike; H(2)O(2)-degrading activity of cell lysates was elevated at ratios of 10 and higher. REN/PECAM cells internalize 66% of cell-bound anti-PECAM/(125)I-catalase. Confocal microscopy localized anti-PECAM/catalase to intracellular vesicles, while catalase expressed by adenovirus was distributed in vesicles and throughout the cytosol. Within 15 min of delivery, anti-PECAM/catalase augmented H(2)O(2)-degrading activity and survival of H(2)O(2)-exposed REN/PECAM cells. The effects of conjugate delivery reached a plateau within 1 h and declined to the basal level within 12 h. In contrast, adenoviral delivery required several hours for transduction and development of the effects, but permitted much longer duration of protection (at least 48 h). Simultaneous exposure of REN/PECAM cells to anti-PECAM/catalase and catalase-encoding adenovirus afforded protection against H(2)O(2) with a rapid onset and a prolonged duration. Therefore, PECAM-directed immunotargeting provides a specific, antigen-directed intracellular delivery of catalase that affords a rapid but transient protection against H(2)O(2) and may complement gene delivery strategies for antioxidant protection.