Male chickens, Gallus gallus, produce qualitatively different alarm calls in response to terrestrial and aerial predators. Field observations suggest that ground alarm calls are evoked principally by animals moving on the substrate, while aerial alarm calls are produced in response to objects moving overhead. In the present study, both types of alarm call were elicited in the laboratory using visual stimuli presented on a video-monitor. Aerial alarm calls were reliably evoked by computer-generated animations simulating a soaring raptor; ground alarm calls were produced in response to a 60-s video recording of a raccoon. Playback experiments were then conducted with six exemplars of each call type, selected to represent the natural acoustic variation in both spectral characteristics and duration. All of the test sessions were videotaped and then analysed, frame-by-frame, by a 'blind' observer. Following playback of aerial alarm calls, hens were significantly more likely to run toward an area of cover than after ground alarm calls or a background noise control. Aerial alarm calls evoked crouching, whereas ground alarm calls caused hens to adopt an erect 'vigilant' posture. Both call types increased the rate of horizontal scanning, but hens were significantly more likely to look upward following presentations of aerial alarm calls. These data demonstrate that chicken alarm calls are functionally referential, encoding sufficient information about the circumstances of production for conspecifics to respond appropriately, even in the absence of contextual cues potentially provided by the non-vocal behaviour of the sender.