We have characterized the binding of [125I-iodo-histidyl, methyl Phe7]neurokinin B (125I-NKB) to the human neurokinin-3 (NK3) receptor. 125I-NKB specifically binds to the NK3 receptor expressed in CHO cells with a Kd of 0.2 nM. The ligand displays little crossreactivity with the human NK1 and NK2 receptors. The binding of 125I-NKB to the human NK3 receptor and to rat cortex membranes is inhibited by neurokinin B with IC50 of 1.5 nM and 4 nM, respectively. In contrast, 350- to 500-fold higher concentrations of substance P and neurokinin A are required to inhibit binding to either receptor preparation. The data suggest that 125I-NKB is a high affinity, selective ligand for the human and rat NK3 receptor.