With the persistent threat of emerging infectious diseases and bioterrorism, it has become increasingly important that clinicians be able to identify the diseases that might signal the occurrence of these unusual events. Essential to a thoughtful diagnostic approach is understanding when to initiate a public health investigation and how to appropriately use commonly performed microbiology procedures in the sentinel laboratory to evaluate potential pathogens. Although diagnostic test development is evolving rapidly, recognizing many of these pathogens continues to challenge the capabilities of most sentinel laboratories. Therefore, effective, ongoing communication and education among clinicians, infection control personnel, sentinel laboratorians, public health authorities, and Laboratory Response Network reference laboratorians is the key to preparedness.