Single-chain mouse Kd molecules (SC-Kd) were engineered by connecting residue 276 of Kd heavy chain to the first residue of beta 2-microglobulin through spacers of various lengths, and expressed intracellularly in monkey COS-1 cells. Labeled SC-Kd molecules were found to react with several monoclonal antibodies which recognize native Kd molecules. SC-Kd-15 (with a spacer of 15 residues) was studied in more details. It could be purified and shown to regain a native-like structure after treatment with denaturing agents. Purified SC-Kd-15 could bind certain peptides in a manner qualitatively similar to the Kd.