Translation initiation in eukaryotes is a complex and highly regulated process requiring the action of at least 12 protein factors. The pathway is distinguished by the formation of a pre-initiation complex that recruits the 5' end of the mRNA and scans along it to locate the start codon. During the past decade, a combination of genetics, biochemistry and structural studies has begun to illuminate key molecular events in this critical phase of gene expression. Here, we outline our current understanding of eukaryotic translation initiation and discuss important outstanding challenges.