The adenovirus major late promoter (MLP) has played a pre-eminent role in the analysis of transcription initiation in mammalian cells, and is an outstanding example of the ways in which the study of adenovirus has led to fundamental insights into general cellular processes. The aim of this chapter is to give a comprehensive review of the structure and function of this model mammalian promoter. After a brief description of late transcription in the adenovirus replication cycle, the experimental evidence for the current consensus on the genetic structure of the MLP, including a consideration of non-primate adenovirus MLPs, will be reviewed. Next, the functions of the MLP in the viral life cycle will be examined, and some of the problems that remain to be resolved will be addressed. The review ends with some ideas on how the knowledge of the structure and function of the MLP can be used in designing virus vectors for specific experimental purposes.