The intratracheal (IT) injection technique has been widely used in the mouse studies of pulmonary diseases. Here, we describe a non-invasive technique using oral instillation challenge with the surfactant phospholipid that may advantageously replace the traditional IT technique. We performed comparative studies between oral instillation and IT injection of both vectors (adeno-associated virus, AAV vector) and bacteria (Pseudomonas aeruginosa). Our results demonstrated that the oral instillation is a reliable alternative to IT injection. The administration of a fluorophore-labelled AAV vector demonstrated a similar pattern of distribution and quantity of vector delivered by oral instillation compared with IT injection. In addition, administration of AAV5-alpha-1 antitrypsin (AAT) to the lungs by oral instillation resulted in similar levels of AAT in both the lung homogenates and sera compared with the IT injection group. In our study of P. aeruginosa delivery, oral instillation resulted in similar mouse weight loss, cytokine levels in the epithelial lining fluid [interleukin (IL)-1β, IL-6, tumour necrosis factor-α, neutrophil chemokine and macrophage inflammatory protein-1α], lung histology/pathology and bacterial loads. Therefore, we conclude that oral instillation of materials mixed with surfactant phospholipid is an adequate and reproducible technique to replace the invasive IT injection procedure for the delivery of either vector or bacteria to the lungs. This procedure has the benefits of eliminating the discomfort, local inflammation and mortality associated with the more invasive IT surgical procedures. © Laboratory Animals Ltd.