The role of various essential components in mammalian cell protein chain initiation, such as the 40S ribosomal subunit and the initiation factors, have been studied largely in fractionated systems and little is known about the mechanisms of their regulation. The process was studied in an unfractionated system derived from temperature-sensitive (ts) 422E cells. The mutant cells fail to assemble 60S ribosomal subunits at the nonpermissive temperature with little effect on the production of 40S ribosomal subunits. Although the resulting imbalance in ribosomal subunits has little immediate effect on the overall rate of protein synthesis, the nascent 40S subunits in mutant cells appear to be markedly unstable at the nonpermissive temperature. The initial step in protein synthesis, initiation factor elF-2-promoted binding of [35S]Met-tRNAf Met to 40S ribosomal subunits, remains unimparied in ts 422E cells at the nonpermissive temperature. However, most of the nascent 40S ribosomal subunits in ts 422E cells band at a higher density, suggesting their failure to bind initiation factor elF-3. These newly synthesized 40S subunits in the mutant cells are unstable, and may be degraded before their appearance in the polysomes.