Vaults are cytoplasmic ribonucleoprotein structures that display a complex morphology reminiscent of the multiple arches which form cathedral vaults, hence their name. Previous studies on rat liver vaults (Kedersha, N. L., and L. H. Rome. 1986. J. Cell Biol. 103:699-709) have established that their composition is unlike that of any known class of RNA-containing particles in that they contain multiple copies of a unique small RNA and more than 50 copies of a single polypeptide of 104,000 Mr. We now report on the isolation of vaults from numerous species and show that vaults appear to be ubiquitous among eukaryotes, including mammals, amphibians (Rana catesbeiana and Xenopus laevis), avians (Gallus Gallus), and the lower eukaryote Dictyostelium discoideum. Electron microscopy reveals that vaults purified from these diverse species are similar both in their dimensions and morphology. The vaults from these various species are also similar in their polypeptide composition; each being composed of a major polypeptide with an approximate mass of 100 kD and several minor polypeptides with molecular masses similar to those seen in the rat. Antibodies raised against rat vaults recognize the major vault protein of all species including Dictyostelium. Vaults therefore appear to be strongly conserved and broadly distributed, suggesting that their function is essential to eukaryotic cells.