The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. Although its precise function is unknown, PrPC represents the substrate for the generation of a conformational pathogenic isoform (PrPSc) in human and animal transmissible spongiform encephalopathies, or prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrPC by immunoblot of two-dimensional (2-D) gel electrophoresis preparations, using specific antibodies. Here, we show that PrPC from brain and CSF is composed of several charge isomers of differently glycosylated isoforms of the full-length PrPC and two N-terminally truncated fragments of 20 and 18 kDa. In the CSF, substantial amounts of the highly glycosylated PrPC isoforms and of the unglycosylated 18 kDa fragment are detected. Our study, for the first time, provides a detailed 2-D map of human PrPC both in brain and CSF, and establishes an innovative and sensitive method that might help in detecting the CSF pathological PrPSc isoform in vivo. It also shows the incredible microheterogeneity of such isoforms (ca. 60 spots!), as revealed in 2-D mapping, as opposed to 3-4 main zones by mono-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE).