Aims: We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis. Methods and results: The viability and infectivity of the challenge suspension was determined in relation to aerosolization time, concentration, method of preparation and M. tuberculosis strain. Challenge stocks generated from frozen aliquots of M. tuberculosis were shown to undergo a 1log10CFUml-1 decrease in viability during the first 10min of aerosolization. This correlated with a decrease in surface lung lesions developing in guinea pigs challenged during this time. The phenomenon of decreased viability in vitro was not observed when using freshly grown, nonfrozen cells of M. tuberculosis. The viability of aerosolized bacilli at the point of inhalation relative to the point of aerosolization always remained constant. Conclusion: Based on these findings, we have developed an improved strategy by which to reproducibly deliver aerosol infection doses to individually challenged animals and separately challenged groups of animals. Significance and Impact of the Study: Study of the aerobiological characteristics of micro-organisms is a critical step in the validation of methodology for aerosol infection animal models, particularly where large numbers of animals and nonhuman primates are used.