Prion protein (PrP(C)) is the normal isoform of PrP(Sc), a protein involved in neurodegenerative disorders. PrP(C) participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress-inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP(C) mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP(C), STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP(C), in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP(C) ligands, their expression was not altered in PrP(C)-null mice.