Transcriptional initiator (Inr) elements, like TATA boxes, are found in the core promoters of many eukaryotic protein-coding genes. To facilitate the dissection of transcription initiation mechanisms, and to identify Inr elements within known and newly-discovered genes, the precise DNA sequence requirements for Inr activity must be defined. Previously, we reported a preliminary Inr consensus sequence based on an extensive mutant analysis carried out in HeLa cell extracts. This analysis was limited, however, because it was performed only with an in vitro transcription assay and with the mutants in only one promoter context. In this study, we have assessed the general validity of the functional Inr consensus sequence by analyzing a selected set of mutants in additional promoter contexts and with both in vivo and in vitro assays. In addition, we analyzed the evolutionary conservation of the Inr consensus sequence by determining the sequence requirements for Inr function in Drosophila embryo extracts. The results demonstrate the generality and strict conservation among vertebrates and invertebrates of the Inr consensus sequence, Py Py A+1 N T/A Py Py.