Recently, a novel high-affinity peptide antagonist, 1229U91, was published as a selective neuropeptide Y Y1 antagonist. The selectivity of 1229U91 was evaluated in the human NPY Y1 receptor containing cell line, SK-N-MC, and cells containing the cloned human NPY Y2, the pancreatic polypeptide-preferring (NPY Y4), and the NPY Y5 receptors. 1229U91 potently displaced [125I]-peptide YY (PYY) binding to human NPY Y1 receptors (IC50 = 0.245+/-0.004 nM, n = 4). but displayed little affinity for the human NPY Y2 and Y5 receptors (IC50 > 1000 nM). Interestingly, 1229U91 displaced [125I]-PYY with even greater affinity at the human NPY Y4 receptor (IC50 = 0.081+/-0.009 nM, n = 4). Using a cyclic AMP accumulation assay, 1229U91 blocked NPY inhibition of forskolin-induced adenylate cyclase activity in NPY Y1 receptor containing SK-N-MC cells. In the human NPY Y4 receptor expressing cell line, 1229U91 did not block pancreatic polypeptide (PP) inhibition of forskolin stimulated adenylate cyclase. However, in the absence of PP, 1229U91 was able to inhibit forskolin stimulated cyclic AMP accumulation (IC50 = 7.16+/-2.8 nM, n = 4). We conclude that 1229U91 binds non-selectively with high affinity to both human NPY Y1 and Y4 receptors. Furthermore, 1229U91 displays antagonist activity at the NPY Y1 receptor, while having agonist activity at the NPY Y4 receptor.