During vertebrate central nervous system development, the apical neuroepithelium is bathed with embryonic Cerebrospinal Fluid (e-CSF) which plays regulatory roles in cortical cell proliferation and maintenance. Here, we report the first proteomic analysis of human e-CSF and compare it to an extensive proteomic analysis of rat e-CSF. As expected, we identified a large collection of protease inhibitors, extracellular matrix proteins, and transport proteins in CSF. However, we also found a surprising suite of signaling and intracellular proteins not predicted by previous proteomic analysis. Some of the intracellular proteins are likely to represent the contents of microvesicles recently described within the CSF (Marzesco, A. M., et al. J. Cell Sci. 2005, 118 (Pt. 13), 2849-2858). Defining the rich composition of e-CSF will enable a greater understanding of its concerted actions during critical stages of brain development.