Substantial similarities and profound differences mark ribosomes across phylogeny. The ribosomal core is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. This difference in size is predominantly due to the extension of specific rRNA regions called expansion segments. Here, we characterize expansion segment 7 of the 28S rRNA from Homo sapiens by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. Our results indicate that, in a cell-free environment, G-quadruplexes form in human ES7, or 28S rRNA, and in 80S ribosomes or polysomes purified from human cell lines. rRNA G-quadruplex regions are preferentially located on the most surface-exposed regions of the ribosome, near the termini of specific rRNA tentacles in ES7 and ES27. By multiple sequence alignments, we have inferred that G-quadruplex-forming sequences appear to be a general feature of the surfaces of ribosomes of the phylum Chordata. In this study, we also identify the proteins that bind selectively to the rRNA G-quadruplexes, which include several RNA helicases and other RNA remodeling proteins. It is known that G-quadruplexes are contained in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported to form in ribosomes.