Two 30 S ribosomal proteins from Escherichia coli have been found to be uniquely required for assembly of 30 S ribosomal subunits. 30 S ribosomes were reconstituted in vitro from 16 S RNA and a mixture of purified 30 S ribosomal proteins (sigma Si). In the absence of S16, sigmaSi-S16 particles were slowly assembled wich had physical and functional properties similar to complete particles (sigmaSi). The results indicate that S16 affects the rate of 30 S ribosome assembly, but does not appear to be directly involved in any known ribosomal function. Particles assembled in the absence of S18 (sigmaSi-S18) had high activity in poly(U)-directed polyphenylalanine synthesis but lost considerable activity upon isolation or purification. The loss of activity could be attributed primarily to the loss of proteins S11 and S21. S18 appears to have a major role in the stabilization of ribosome structure, especially the binding of proteins S11 and S21, and does not appear to be directly required for activity in poly(U)-directed polyphenylalanine synthesis. However, it is possible that S18 has some functional role which is not required for polyphenylalanine synthesis.