Recently published models of the Escherichia coli 70 S ribosome at 20 A resolution, obtained by cryo-electron microscopy (cryo-EM) combined with computerized image processing techniques, exhibit two features that are directly relevant to the in situ three-dimensional folding of the rRNA molecules. First, at this level of resolution many fine structural details are visible, a number of them having dimensions comparable to those of nucleic acid helices. Second, in reconstructions of ribosomes in the pre- and post-translocational states, density can be seen that corresponds directly to the A and P site tRNAs, and to the P and E site tRNAs, respectively, thus enabling the decoding region on the 30 S subunit to be located rather precisely. Accordingly, we have refined our previous model for the 16 S rRNA, based on biochemical evidence, by fitting it to the cryo-EM contour of ribosomes carrying A and P site tRNAs. For this purpose, the most immediately relevant evidence consists of new site-directed cross-linking data in the decoding region, which define sets of contacts between the 16 S rRNA and mRNA, or between 16 S rRNA and tRNA at the A, P and E sites; these contact sites can be correlated directly with the tRNA positions in the EM structure. The model is extended to other parts of the 16 S molecule by fitting individual elements of the well-established secondary structure of the 16 S rRNA into the appropriate fine structural elements of the EM contour, at the same time taking into account other data used in the previous model, such as intra-RNA cross-links within the 16 S rRNA itself. The large body of available RNA-protein cross-linking and foot-printing data is also considered in the model, in order to correlate the rRNA folding with the known distribution of the 30 S ribosomal proteins as determined by neutron scattering and immuno-electron microscopy. The great majority of the biochemical data points involve single-stranded regions of the rRNA, and therefore, in contrast to most previous models, the single-stranded regions are included in our structure, with the help of a specially developed modelling programme, ERNA-3D. This allows the various biochemical data sets to be displayed directly, in this and in the accompanying papers, on diagrams of appropriate parts of the rRNA structure within the cryo-EM contour.