For the determination of prostaglandin (PG) D 2 produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD 2 is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD 2. In an attempt to get a specific antibody for PGD 2, we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD 2, a novel, chemically stable, isosteric analogue of PGD 2. We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD 2. To develop the enzyme-linked immunosorbent assay (ELISA) for PGD 2, the immobilized antigen using the stable PGD 2 derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD 2. The optimization of the assay provided a sensitive calibration curve for PGD 2 from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD 2 was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD 2 in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD 2 in the maturation medium of adipocytes at 37 °C caused the chemical conversion into PGJ 2 derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD 2 before the conversion starts to occur.