A panel of anti-rhodopsin monoclonal antibodies (MAbs) of defined epitope specificity has been evaluated by immunocytochemistry. Most of the IgG class MAbs (23/27) gave positive results, but only a few of the IgM class MAbs (2/21) were useful for this application. MAbs specific to the N-terminal region stained rod outer segments most strongly, with progressively less staining in the Golgi, perikarya, plasma membrane of the inner segment, and synaptic region. Phagosomes located basally in the pigment epithelium were stained; cone cells were consistently negative. Antibodies to the C-terminus of rhodopsin labeled the same cell structures (except for phagosomes) but showed diversity in their binding pattern. Many of these MAbs bound to cone outer segments in addition to rods, and showed different patterns of binding to red/green and blue cones. Antibodies specific for rhodopsin sequence 340-348 labeled different types of cone cells, indicating differences in their binding sites. Two MAbs were found to label hydrophilic loop sequences which connect rhodopsin's transmembrane segment: MAb K42-41 which binds loop 5-6, and MAb A1-55 which binds loop 2-3. At least these two regions of the rhodopsin sequence in addition to the C- and N-termini, are available for antibody reaction in fixed retina.