Faecal samples, collected from 200 healthy animals in Antwerp Zoo, were examined for the presence of pathogenic Listeria spp. A two-stage standard isolation (ISO) method was combined with immunomagnetic separation (IMS). ALOA agar, a chromogenic isolation medium, differentiating Listeria spp. on the basis of beta-glucosidase and phosphatidylinositol-specific phospholipase C (PIPLC) activity, was compared with PALCAM agar. Confirmation of the isolates was based on conventional biochemical tests and a disc test, which detects a specific aminopeptidase produced by all Listeria spp. except Listeria monocytogenes. Listeria spp. were isolated from 42 (21.0%), L. monocytogenes from 14 (7.0%), and Listeria ivanovii from two (1.0%) faecal samples. The application of IMS after primary enrichment detected pathogenic Listeria spp. in 12 (6.0%) samples. The ISO method, combining primary and secondary enrichment, detected pathogenic Listeria spp. in 15 (7.5%) samples. The sensitivity of IMS compared to the ISO method was 73.3% and the specificity was 99.5%. ALOA agar was superior to PALCAM agar for isolation of Listeria spp. The disc test identified all L. monocytogenes isolates. IMS after primary enrichment was a suitable screening method, but secondary enrichment increased the number of positive samples.