Inhibition of return (IOR) is the reduction of detection speed and/or detection accuracy of a target in a recently attended location. This phenomenon, which has been discovered and studied thoroughly in humans, is believed to reflect a brain mechanism for controlling the allocation of spatial attention in a manner that enhances efficient search. Findings showing that IOR is robust, apparent at a very early age and seemingly dependent on midbrain activity suggest that IOR is a universal attentional mechanism in vertebrates. However, studies in non-mammalian species are scarce. To explore this hypothesis comparatively, we tested for IOR in barn owls (Tyto alba) using the classical Posner cueing paradigm. Two barn owls were trained to initiate a trial by fixating on the center of a computer screen and then turning their gaze to the location of a target. A short, non-informative cue appeared before the target, either at a location predicting the target (valid) or a location not predicting the target (invalid). In one barn owl, the response times (RT) to the valid targets compared to the invalid targets shifted from facilitation (lower RTs) to inhibition (higher RTs) when increasing the time lag between the cue and the target. The second owl mostly failed to maintain fixation and responded to the cue before the target onset. However, when including in the analysis only the trials in which the owl maintained fixation, an inhibition in the valid trials could be detected. To search for the neural correlates of IOR, we recorded multiunit responses in the optic tectum (OT) of four head-fixed owls passively viewing a cueing paradigm as in the behavioral experiments. At short cue to target lags (<100 ms), neural responses to the target in the receptive field (RF) were usually enhanced if the cue appeared earlier inside the RF (valid) and were suppressed if the cue appeared earlier outside the RF (invalid). This was reversed at longer lags: neural responses were suppressed in the valid conditions and were unaffected in the invalid conditions. The findings support the notion that IOR is a basic mechanism in the evolution of vertebrate behavior and suggest that the effect appears as a result of the interaction between lateral and forward inhibition in the tectal circuitry.