To identify molecular interaction partners of the cellular prion protein (PrP^C), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP^C. Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP^C in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise β-strands C and C′ within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144–154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM^−/−) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (±4.1, SEM) days, arguing that N-CAM is not involved in PrP^Sc replication. Our findings raise the possibility that N-CAM may join with PrP^C in carrying out some as yet unidentified physiologic cellular function.