Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), initiator fMet-tRNAfMet, three initiation factors, IF1, IF2, and IF3, and the large ribosomal subunit (50S). During initiation, the 30S subunit, in a complex with IF3, binds mRNA, IF1, IF2·GTP, and fMet-tRNAfMet to form a 30S initiation complex which then recruits the 50S subunit to yield a 70S initiation complex, while the initiation factors are released. Here we describe a transient kinetic approach to study the timing of elemental steps of 30S initiation complex formation, 50S subunit joining, and the dissociation of the initiation factors from the 70S initiation complex. Labeling of ribosomal subunits, fMet-tRNAfMet, mRNA, and initiation factors with fluorescent reporter groups allows for the direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer (FRET) between two fluorophores. Subunit joining was monitored by light scattering or by FRET between dyes attached to the ribosomal subunits. The kinetics of chemical steps, that is, GTP hydrolysis by IF2 and peptide bond formation following the binding of aminoacyl-tRNA to the 70S initiation complex, were measured by the quench-flow technique. The methods described here are based on results obtained with initiation components from Escherichia coli but can be adopted for mechanistic studies of initiation in other prokaryotic or eukaryotic systems. © 2007 Elsevier Inc. All rights reserved.