Conditions are described for using a primer extension inhibition (toeprinting) assay to study the initiation step of protein synthesis in rabbit reticulocyte lysates. These studies revealed that chloramphenicol acetyltransferase mRNA, which is widely used as a reporter, forms unusually labile initiation complexes. This and other unexpected problems were solved by adjustments in pH and temperature during the reverse transcriptase step. Complications that may occur during the ribosome binding step were also examined, including the possibility of rapid mRNA degradation. The suitability of inhibitors commonly used to block the elongation phase of translation was studied. The refined toeprinting assay was used to confirm context-dependent selection of the AUG start codon. Absence of the m7G cap did not subvert the process wherein initiation is restricted to the first AUG codon. The fidelity of initiation was impaired, however, when NaF was introduced during the ribosome binding step. In a preliminary assessment of the processivity of scanning, no dissociation of 40S ribosomal subunits was detected as the distance from the cap to the AUG codon was increased to nearly 300 bases. With an mRNA that contains a pseudoknot upstream from the AUG codon, the toeprinting assay revealed 40S ribosomal subunits trapped behind the base paired structure. Thus the assay is usable for mapping some intermediates as well as for detecting conventional 80S initiation complexes.