The distribution of neuropeptide Y Y1 and Y2 receptors has been extensively studied in the rat using selective masking of [125I]peptide YY binding by Y1- and Y2-selective peptides. In the present study, we characterized the binding of the subtype-selective radioligands [125I](Leu31-Pro34)-peptide YY and [125I]peptide YY3-36 to rat and guinea-pig brains and identified differences in Y1 and Y2 distribution. [125I](Leu31Pro34)-peptide YY and [125I]peptide YY3-36 bound to single sites in rat and guinea-pig brains with pharmacologies consistent with the Y1 and Y2 receptors, respectively. Autoradiographic studies were conducted using adjacent sections from rat and guinea-pig brains. Compared to the rat, the distribution of both Y1 and Y2 receptors was markedly different in the guinea-pig. For example, a high level of Y1 binding was detected within the thalamus of the rat while, in the guinea-pig, very little Y1 binding was observed in this region. Y1 binding was very low in the rat hippocampus, while the guinea-pig hippocampus contained high levels of Y1 binding. High levels of both Y1 and Y2 binding were observed in the guinea-pig cerebellum while, in the rat, only low levels of Y2 binding were visible. Therefore, the guinea-pig brain exhibits dramatic differences in the pattern of Y1 and Y2 receptors when compared to the rat. It is likely that Y1 and Y2 receptors perform different roles in the central nervous system of guinea-pigs and rats.