Proper temperature control is essential in preventing Clostridium perfringens food poisoning. The U.S. Department of Agriculture Food Safety and Inspection Service cooling guidelines offer two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log CFU/g increase of C. perfringens and no growth of Clostridium botulinum. The latter option requires laboratory challenge studies to validate the efficacy of a given cooling process. Accordingly, the objective of this study was to investigate the role of several methodological variables that might be encountered during typical C. perfringens challenge studies. Variables studied included plastic bag type (Whirlpak or Spiral Biotech), sealing method (Multivac or FoodSaver), initial spore inoculum size (1 to approximately 3 log CFU/g), and growth environment (ground beef or Trypticase-peptone-glucose-yeast extract [TPGY] broth). The major factors that affected growth were sample bag type and growth environment. Samples incubated in Whirlpak bags showed significantly less growth than those incubated in Spiral Biotech bags, which was likely due to the former bag's greater oxygen permeability. C. perfringens spores showed shorter germination, outgrowth, and lag times and C. perfringens cells showed faster growth rates in ground beef compared with TPGY broth. No significant difference was observed between two different sealing methods. Initial spore inoculum levels in the range studied had no significant effect on final C. perfringens cell concentration.