An auto-anti-idiotypic approach was used to generate mouse monoclonal anti-idiotypic antibody E9F11F6 (Ab2) to progesterone. A conjugate of 4-pregnane 3,20 dione and bovine serum albumin was used as the immunogen. E9F11F6 bound to a rabbit anti-progesterone antibody in a linear concentration-dependent manner. It inhibited the binding of [3H]progesterone to a monoclonal anti-progesterone antibody, E9D5 (Ab1); this inhibition was concentration dependent. Results from chase experiments showed that this inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the ligand combining site. Indirect immunofluorescence studies also showed that Ab2 and progesterone bound to similar binding sites on Ab1. Overall, the results suggest that E9F11F6 contains an internal image of at least part of the progesterone molecule, and that it can be classified as an Ab2 beta antibody. This anti-idiotypic antibody may be of value in further studies of endocrine and reproductive physiology.