Background: Aerosol delivery through an endotracheal tube during mechanical ventilation of small animals, simulating neonates and small infants, has shown to be influenced by a variety of factors including aerosol generator type, droplet/particle size, ventilator circuitry and ventilation regime. A review of the literature indicates that reported aerosol deposition rates in rodents are quite low, with lung deposition in anesthetized, mechanically ventilated rats reported to be similar to 3.9 and similar to 8% in anesthetized, spontaneously breathing rats. The optimization of aerosol delivery to both in vitro and in vivo models of anesthetized mechanically ventilated rodents is described in this study.Methods: Characterization and optimization of the in vitro system performance relied on gravimetric analysis, laser diffraction droplet sizing, and spectrophotometric analysis of drug mass on inspiratory filters. The optimized setup was subsequently employed in vivo to determine deposition of a tracer aerosol in the rat lung.Results: In vitro testing confirmed that droplet size, ventilation regimen, breath actuation setting, and the inclusion of a drug recycling step had the greatest effect on inhaled mass. During testing, improvements of up to 41% were seen in inhaled mass values between runs with the addition of a recycling step. The negative effects of the aerosolization process on albuterol sulphate were minimal. In vitro deposition rates of 29.95 +/- 1.54% of the original dose were recorded (n = 3). In vivo deposition rates of Evans blue were highly comparable (30.88 +/- 5.73%) (n = 6). Intratracheal instillation of the tracer dye resulted in deposition of 87.34 +/- 6.23% of the original dose.Conclusions: This optimized experimental setup allows for greater inhaled mass than previously reported. The addition of a recycling step may prove to be a significant improvement in achieving higher deposition in mechanically ventilated lungs; however, the suitability of the test agent for repeated nebulization needs assessment.