Respiratory failure is a significant problem within the pediatric population. A means of respiratory support that readily allows ambulation could improve treatment. The Pittsburgh Pediatric Ambulatory Lung (P-PAL) is being developed as a wearable pediatric pump-lung for long-term respiratory support and has previously demonstrated positive benchtop results. This study aimed to evaluate acute (4-6 hours) in vivo P-PAL performance, as well as develop an optimal implant strategy for future long-term studies. The P-PAL was connected to healthy sheep (n = 6, 23-32 kg) via cannulation of the right atrium and pulmonary artery. Plasma-free hemoglobin (PfHb) and animal hemodynamics were measured throughout the study. Oxygen transfer rates were measured at blood flows of 1-2.5 L/min. All animals survived the complete study duration with no device exchanges. Flow limitation because of venous cannula occlusion occurred in trial 2 and was remedied via an altered cannulation approach. Blood exiting the P-PAL had 100% oxygen saturation with the exception of trial 4 during which inadequate device priming led to intrabundle clot formation. Plasma-free hemoglobin remained low (<20 mg/dl) for all trials. In conclusion, this study demonstrated successful performance of the P-PAL in an acute setting and established the necessary methods for future long-term evaluation.