Tuberculosis continues to be a major world health threat. The etiologic agent is among the vegetative organisms most resistant to chemical disinfection. Tuberculocidal efficacy testing for regulatory approval of chemical germicides has evolved considerably over the past decade. A method currently in use is the Environmental Protection Agency Tuberculocidal Activity Test Method, a suspension test using a Mycobacterium bovis culture grown under specific conditions and stored frozen until used. Differing tuberculocidal label claims on products with similar formulations have raised questions concerning the equivalence of test suspensions prepared by different laboratories. Five M. bovis suspensions from laboratories currently performing this test were compared against a battery of three disinfectants at a single test site. A significant difference between test cultures was found, with two of the five exhibiting a significant difference from the other three and also from each other. There was a significant culture-by-disinfectant interaction, indicating that the five cultures did not respond in a consistent manner across the different disinfectants used. However, these differences were due to cultures that were not prepared in accordance with the standard procedure or otherwise did not meet the test suspension criteria. In addition, a 0.55% sodium hypochlorite solution was found to be a sensitive indicator of culture variability. These data reinforce the need to adhere to published procedures and guidelines when growing and preparing a tuberculocidal test suspension and shed light on the variables associated with this type of testing.