Heightened interest in pathogens with the potential for aerosol transmission and for which prevention and medical treatment is not readily available has resulted in a need for more work environments that meet Biosafety Level 3 (BSL 3) criteria. Recognizing that the facility-based criteria for BSL 3 cannot be achieved by some existing laboratories, the Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH) biological safety guidelines provide an option for attaining BSL 3 status through the use of Biosafety Level 2 (BSL 2) facilities and strict adherence to BSL 3 practices (BSL 2/3). Inherent to this provision is a greater emphasis on safe work practices. Since the extent to which this approach is actually used in practice is not known, a nationwide mail survey of medical academic and research institutions was conducted to provide an objective indication of the proportion of BSL 3 operations actually being carried out in the BSL 2/3 mode. The results obtained indicate that 2% of activities designated as BSL 3 in the study population actually achieve this level of protection using the BSL 2/3 approach. The findings quantitatively estimate for the first time the proportion of BSL 3 activities being carried out in this fashion, and can serve as a reference point for future studies to evaluate usage trends. The results also demonstrate the utility of flexible, performance-based health and safety guidelines, as a significant amount of clinical and research work is being accommodated with the BSL 2/3 provision.