Neuropeptide Y (NPY) in the CNS plays an important regulatory role in anxiety-related responses as exogenous administration of NPY exerts an anxiolytic-like effect in rodents. This effect is believed to be mediated by the Y(1) receptor system as pharmacological modulation of this Y(1) receptor system results in an increase in anxiety. Here we present a comprehensive phenotyping strategy for characterizing Y(1) receptor knockout animals at different times of the circadian rhythm using several motor activity-, exploration-, and anxiety-related behavioural tasks including open field, elevated plus maze, light-dark, and hole board test. We show that Y(1) deficiency has an important effect on motor activity and explorative-like behaviours and that it results in marked alterations in anxiety-related behaviours. Importantly, the behavioural phenotype of the Y(1) receptor knockout mice is circadian rhythm-dependent and also influenced by stimuli such as restraint stress. In addition, we found evidence for increases in working memory. Taken together, these findings suggest an important role of Y(1) receptors in the regulation of motor activity, exploration, and anxiety-related behaviours. This role is also influenced by several factors such as circadian rhythm and stress exposure confirming the importance of a comprehensive strategy and of using genetic animal models in behavioural neuroscience.