A panel of twelve monoclonal antibodies (mAbs) to plectin was analyzed to localize molecular epitopes and assess their utility for various immunotechniques. Based on staining patterns obtained by immunoblotting of proteolytic plectin fragments five groups of mAbs with spatially closely linked epitopes were distinguished. In combination with previous results obtained with recombinant mutant proteins, the epitopes of all mAbs could be shown to reside within three separated domains of plectin's rod domain. Immunoblot analyses of several different rat tissues and a number of cultured cell lines derived from various species, including rat, hamster, cow, mouse and man, revealed considerable variations in immunoreactivity of antibodies. Differences in mAb immunoreactivities were also revealed by immunofluorescence microscopy of different tissues and cultured cell lines. One third of the mAbs examined produced staining patterns that were indistinguishable from those generated by conventional rabbit antisera, confirming the widespread and highly divers cellular localization of plectin previously reported. Microinjection of several monoclonal antibodies into Rat 1 cells had no detectable effects on the structure and organization of plectin arrays or of other cytoskeletal filaments. This new collection of mAbs provides a more reliable tool for histological studies than previously available antisera and should be useful for studies of tissue and cell type specific functions of plectin domains in vivo and in vitro.