The fine specificities of two panels of monoclonal antibodies (mAbs) for sheep major histocompatibility complex (MHC) class II molecules were determined using five mouse L-cell transfectants, each expressing a defined sheep DQ or DR MHC class II A/B gene pair. Using the transfectants in an indirect fluorescence antibody assay, previous immunochemical characterization of the mAbs was confirmed for 16 of 23 mAbs tested. The MHC class II subtype specificity (DQ or DR) of each mAb was assigned without interference from the products of other expressed class II loci. This allowed the identification of both cross-locus specificities as well as defining fine specificities of mAbs previously only partially characterized by immunochemical techniques.