OBJECTIVE The Laboratory Response Network (LRN) is the United States' laboratory system for detecting, confirming, and reporting potential bioterrorism agents. The first tier-sentinel laboratories-is composed principally of hospital-based laboratories and is tasked with ruling out potential biological threat agents in clinical specimens or the identification of suspicious specimens for further testing in higher tiers of the LRN system. The aim of the present study was to broadly describe preparedness of the first tier of the hospital LRN, the sentinel laboratories, with a specific focus on training, personnel, and communications. METHODS A semistructured cross-sectional survey of US sentinel laboratories was designed and conducted. Hospitals with greater than 250 beds and an emergency department were considered eligible for inclusion. A geographically weighted sample of 201 hospitals was selected for inclusion. The survey was administered by telephone to the microbiology managers (or designees) at the selected hospitals. The survey contained questions related to drill frequency, proficiency survey participation, personnel training, personnel responsibilities, procedures for biological threat response, and overall confidence in preparedness. RESULTS Overall, 179 hospitals (89.1%) identified themselves as sentinel laboratories and participated in the survey; 11.7% reported that they had had an emergency alert within the last 2 years. Although rates of internal drills were low (20.7%), participation in some form of bioterrorism proficiency evaluation was high (79.9%). In all, 83.8% of laboratories reported that they had personnel designated to coordinate response to acts of bioterrorism. More than 73% of respondents indicated that they had sufficient personnel, equipment, and training to respond to a biological terrorism event. By multivariate analysis, sentinel laboratories were 3.4 times more likely to feel confident that they had sufficient personnel, equipment, and training to respond to a biological terrorism event if they had designated personnel for bioterrorism roles. CONCLUSIONS This pilot study of sentinel laboratory bioterrorism preparedness demonstrated that hospital laboratory personnel, training, and communication preparedness were not universal, despite designation as sentinel laboratories. A need for unified monitoring of sentinel laboratories exists, and efforts should be made to develop standardized metrics for sentinel laboratory preparedness.