The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.