In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, 125I‐BH‐[Leu15]‐gastrin‐(5‐17) [(3‐[125I]iodo‐4‐hydroxyphenyl)‐propionyl‐[Leu15]‐gastrin‐(5‐17)]. Binding of 125I‐BH‐[Leu15]‐gastrin‐(5‐17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and 125I‐BH‐CCK‐8 [(3‐[125I]iodo‐4‐hydroxyphenyl)‐propionyl‐CCK‐8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 ± 900 binding sites/cell (Kd = 0.52 ± 0.23 nM) and 6200 ± 1100 binding sites/cell (Kd= 0.31 ± 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding correlated well with those obtained using 125I‐BH‐CCK‐8. Selective CCK/gastrin antagonists L‐364,718 (MK‐329) and L‐365,260 also inhibited 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding. These results indicate that 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK‐B/gastrin receptor. Saturation experiments have shown that both 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and 125I‐BH‐CCK‐8 interact with a single class of high‐affinity binding sites in the Jurkat cell line. Binding characteristics of 125I‐BH‐[Leu15]‐Gastrin‐(5‐17) and 125I‐BH‐CCK‐8 were estimated to be about 2500 ± 400 binding sites/cell (Kd= 0.19 ± 0.09 nM) and 2400 ± 350 binding sites/cell (Kd= 0.06 ± 0.02 nM), respectively. Furthermore, the apparent affinities of CCK analogs and selective antagonists MK‐329 and L‐365,260 for the sites labeled by both probes were identical. The biological activity of cold 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and [Leu15]‐gastrin‐(5‐17) was demonstrated by their ability to increase intracellular calcium concentration in Jurkat cells. All these experiments showed that 125I‐BH‐[Leu15]‐gastrin‐(5‐17) provides a convenient ligand for gastrin receptor binding studies. © Munksgaard 1994.