An initial cluster of 2009 influenza A (H1N1) cases among healthcare workers was reported at the Limoges University Hospital (CHU) in August 2009. An epidemiological investigation involving active case finding of cases reported between August 11 and 30 was conducted. Epidemiological and clinical data were collected during structured interviews. Testing for the 2009 influenza A (H1N1) virus was performed using reverse transcriptase-polymerase chain reaction (RT-PCR). A specific questionnaire was used to identify the transmission prevention measures implemented in the affected departments. The investigation identified 9 suspected cases of influenza among healthcare staff in 8 departments of the University Hospital: 4 were classified as confirmed, 2 as probable, and 3 as possible. A link between the first four cases was identified, whereas no epidemiological link was found with the last five cases, suggesting a possible second chain of transmission. Secondary prevention measures, rapidly implemented within the affected departments, included, in particular, the wearing of masks at the first sign of symptoms and work exclusion upon reporting of symptoms. The absence of transmission to patients and the limited transmission among staff working in the same department underscore the importance of strengthening individual and collective prevention measures throughout the hospital shortly after the first case of influenza is reported. (R.A.)