In order to add to the limited data available about the incidence of invasive Listeria monocytogenes infection in the Netherlands, two studies were conducted. In the first study, data on hospital patients with listeriosis in the period 1995-2003 were obtained from the National Medical Registration (study 1). In the second study, hospital discharge letters for patients whose Listeria isolates were received by the Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM) in the period 1999-2003 were retrieved (study 2). Serotyping and pulsed-field gel electrophoresis (PFGE) were used to subtype the various strains of Listeria. These reviews revealed 283 hospital patients and 159 patients with Listeria isolates. Discharge letters were received for 107 (67%) patients. The mean annual incidence of listeriosis in both studies was 2.0 per million inhabitants. The main clinical manifestations were meningitis (incidence: 0.9 and 1.0 per million in studies 1 and 2, respectively) and septicaemia (incidence: 0.08 and 1.0 per million, respectively). Listeriosis in pregnancy was rare (incidence: 1.3 and 2.4 per 100,000 pregnancies over 24 weeks of gestation, respectively). Predisposing conditions were present in 47 and 71% of the patients in studies 1 and 2, respectively. The mortality due to listeriosis was 18%. Serotypes 4b, 1/2a, and 1/2b were responsible for 96% of the cases of human listeriosis. Listeriosis is rare in the Netherlands, but its clinical course is severe and the resulting mortality is high. Therefore, the current recommendations for pregnant women to avoid high-risk foods should be continued. These dietary recommendations should also be given to individuals with predisposing conditions, since they, too, are at risk of Listeria infection.