A selective medium containing polymyxin B, crystal violet, thallous acetate, bacitracin and cycloheximide in 10% sheep blood dextrose starch agar, and a modified Pasteurella multocida-specific polymerase chain reaction (PCR) assay were developed for the respective isolation and detection of P. multocida from chicken alimentary tract. The selective medium and the PCR assay were highly sensitive, detecting 100 cfu from colon contents. These techniques were used to follow the localisation of an orally administered virulent P. multocida in chickens. Pasteurellae could be isolated from the crop of some birds up to 30 h, occasionally from other sites after 28 h. It was concluded that the crop was a likely site for colonisation and that infection was most likely to occur through the mucosa of the jejunum or ileum.