The requirements for the adjustment of fMet-tRNA in the ribosomal P-site have been analyzed by studying the formation of fMet-puromycin in a Bacillus stearothermophilus system. The binding of fMet-tRNA to the 30 S ribosomal subunit is not drastically affected by the omission of GTP, mRNA, mRNA and GTP, or by replacing GTP with GTP analogues. The adjustment of fMet-tRNA in the P site has stricter requirements and fMet-puromycin formation occurred at its maximum rate and extent when fMet-tRNA was bound to 30 S subunits programmed with the AUG triplet or with an mRNA in the presence of GTP. Neither GTP nor the mRNA, however, were found to be essential. Omission of GTP caused only a slight reduction in the rate of fMet-puromycin formation without a significant change of the activation energy, while omission of the template resulted in a requirement for a higher activation energy. In the absence of both GTP and template, however, essentially no fMet-puromycin was formed, indicating that these components cooperate in the adjustment of the initiator tRNA in the P-site. The contribution of various structural elements of the mRNA in determining this adjustment was investigated. It was found that the codon-anticodon interaction and the filling of the ribosomal mRNA channel with a polyribonucleotide are necessary (but not sufficient singly) for the correct orientation of the initiator tRNA in the absence of GTP. The nature of the initiation triplet and the occurrence and/or the strength of the Shine-Dalgarno interaction were also found to contribute to the orientation of the bound fMet-tRNA.