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Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMet-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMet-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2–3-fold preference for fMet-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyi-tRNAs, has a higher affinity for fMet-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2 discriminates in favour of fMet-tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]