By immunizing prion knockout mice (Prnp−/−) with recombinant murine prion protein (PrP^c), we obtained a panel of mAbs specific for murine PrP^c. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrP^c) and protease-resistant (PrP^res) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrP^c. These newly generated PrP^c antibodies will help to explore the biology of PrP^c and to establish the diagnosis of prion diseases in both humans and animals.