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Background Despite the known risk of tuberculosis (TB) to health care workers (HCWs), research suggests that many are not fully adherent with local TB infection control policies. The objective of this exploratory study was to identify factors influencing HCWs' adherence to policies for routine tuberculin skin tests (TSTs) and treatment of latent TB infection (LTBI). Methods Sixteen focus groups were conducted with clinical and nonclinical staff at 2 hospitals and 2 health departments. Participants were segmented by adherence to TST or LTBI treatment policies. In-depth, qualitative analysis was conducted to identify facilitators and barriers to adherence. Results Among all focus groups, common themes included the perception that the TST was mandatory, the belief that conducting TSTs at the work site facilitated adherence, and a general misunderstanding about TB epidemiology and pathogenesis. Adherent groups more commonly mentioned facilitators, such as the perception that periodic tuberculin skin testing was protective and the employee health (EH) provision of support services. Barriers, such as the logistic difficulty in obtaining the TST, the perception that LTBI treatment was harmful, and a distrust of EH, emerged consistently in nonadherent groups. Conclusions This information may be used to develop more effective interventions for promoting HCW adherence to TB prevention policies. Informed efforts can be implemented in coordination with reevaluations of infection control and EH programs that may be prompted by the publication of the revised TB infection control guidelines issued by the Centers for Disease Control and Prevention in 2005.