AbstractSperm uptake of glycosyl phosphatidylinositol (GPI)‐linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as “uterosomes,” in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH‐20), a well‐known hyaluronidase found in male and female fluids, as a model to investigate vesicle‐mediated GPI‐linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be ∼10–70 and ∼15–50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo‐osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle‐mediated GPI‐linked protein transfer to sperm from murine reproductive fluids. Mol. Reprod. Dev. 75: 1627–1636, 2008. © 2008 Wiley‐Liss, Inc.