To maintain rodent colonies free from harmful infectious agents, laboratory animal care programs frequently employ the use of sterilized caging and supplies. Sterilized caging is important for preventing the spread of infectious pathogens from contaminated fomites, for biocontainment, and for safety. We designed several experiments to determine the effects of commonly encountered processes on steam penetration and substrate heat exposure. We used biologic indicators as a proxy for steam penetration. We used the time bedding spent above 121 °C as a proxy for substrate cumulative heat exposure. This temperature was measured using a high-temperature data logger. We first examined the effect of stacking cages with various bedding types on steam penetration. We then autoclaved soiled bedding and studied the variables of bedding type, bagging style, presence of diet and water in the cage, and amount of time between cage change out on steam penetration and cumulative heat exposure. For clean bedding, we found adequate steam penetration regardless of bedding type, cycle program, or location of the cage in the bulk autoclave. For the soiled bedding experiments, there were no differences between bedding types noted. Placement of cages inside plastic bagging increased the amount of time the bedding spent above 121 °C on average but not significantly. There was no difference in steam penetration of bedding or time spent above 121 °C for 2- or 4-wk cage change-out schedules. When cages were autoclaved with diet and water, the time the bedding spent above 121 °C was significantly less than when autoclaving bedding alone, but there was adequate steam penetration for all cages. This study demonstrates that common practices in the industry are effective. Based on the results of this study, it is recommended that each institution evaluate their autoclaving practices and confirm that those practices are sufficient and effective.