A ligand blotting technique was developed to study the HCG-binding protein from Pseudomonas maltophilia after size separation by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were transferred to a nitrocellulose sheet, which was subsequently incubated with [125I]iodo HCG, and subjected to determination for radioactivity in gamma-counter. A radioactivity peak equivalent to an M(r) 70000 appeared, which was not observed when the hormone incubation was performed in the presence of an excess of unlabeled HCG. The peak also disappeared when the protein samples were treated with reducing agent, which showed that integrity disulfide bonds of the protein was essential for the protein-hormone interaction. In addition, position of the radioactivity peak which was due to the binding of [125I]iodo HCG to western blots of the HCG-binding protein was corresponding to that of the antibodies against the HCG-binding protein recognizing a 70000 protein on the western blots. These results show that the HCG-binding protein from Pseudomonas maltophilia is an M(r) 70000 protein and that the HCG-binding protein contains at least one disulfide bond essential to its binding activity.