A fragment of 16S RNA corresponding to most of the 5'-domain (residues 1-526) was prepared by in vitro run-off transcription. When this fragment was incubated with a mixture of 30S proteins under conditions known to result in the in vitro assembly of a complete, functional 30S ribosome from a full-length transcript, a discrete 16S particle was formed. This particle contained near stoichiometric amounts of ribosomal proteins S4, S16, S17, and S20. These four proteins are the same, and only, ones that have been shown to interact with the 5' domain of 16S RNA in the intact 30S ribosome in the footprinting studies of Noller and co-workers. We conclude that the 5' fragment 1-526 is capable of folding independently of the rest of the molecule so as to generate the protein binding sites for the same four proteins with which the corresponding segment of full-length 16S RNA normally interacts. These sites not only include those for S4, S17, and S20 that are known to bind directly to the RNA, but also the site for S16, which requires the prior binding of S4 and S20.