Background: Mesenchymal stromal cells (MSCs) have been demonstrated to attenuate acute lung injury when delivered by intravenous or intratracheal routes. The authors aimed to determine the efficacy of and mechanism of action of intratracheal MSC therapy and to compare their efficacy in enhancing lung repair after ventilation-induced lung injury with intravenous MSC therapy.Methods: After induction of anesthesia, rats were orotracheally intubated and subjected to ventilation-induced lung injury (respiratory rate 18 min(-1), P-insp 35 cm H2O,) to produce severe lung injury. After recovery, animals were randomized to receive: (1) no therapy, n = 4; (2) intratracheal vehicle (phosphate-buffered saline, 300 mu l, n = 8); (3) intratracheal fibroblasts (4 x 106 cells, n = 8); (4) intratracheal MSCs (4 x 106 cells, n = 8); (5) intratracheal conditioned medium (300 mu l, n = 8); or (6) intravenous MSCs (4 x 106 cells, n = 4). The extent of recovery after acute lung injury and the inflammatory response was assessed after 48 h.Results: Intratracheal MSC therapy enhanced repair after ventilation-induced lung injury, improving arterial oxygenation (mean +/- SD, 146 +/- 3.9 vs. 110.8 +/- 21.5 mmHg), restoring lung compliance (1.04 +/- 0.11 vs. 0.83 +/- 0.06 ml.cm H2O-1), reducing total lung water, and decreasing lung inflammation and histologic injury compared with control. Intratracheal MSC therapy attenuated alveolar tumor necrosis factor-alpha (130 +/- 43 vs. 488 +/- 211 pg.ml(-1)) and interleukin-6 concentrations (138 +/- 18 vs. 260 +/- 82 pg.ml(-1)). The efficacy of intratracheal MSCs was comparable with intravenous MSC therapy. Intratracheal MSCs seemed to act via a paracine mechanism, with conditioned MSC medium also enhancing lung repair after injury.Conclusions: Intratracheal MSC therapy enhanced recovery after ventilation-induced lung injury via a paracrine mechanism, and was as effective as intravenous MSC therapy.