Approximately 25% of arthropod RNA polymerase II-transcribed promoters contain one or more copies of the sequence TCAGT beginning within the interval (-10, +10). The clear statistical overrepresentation of this sequence and, to a lesser extent, of its cognates ACAGT, GCAGT, and TCATT, implies that they may be significant promoter elements. Their collective sequence similarity to vertebrate initiators (Inrs) of the TdT class suggests that the vertebrate and arthropod elements are homologous. Prior work in vertebrate systems has emphasized the role of the Inr in promoters lacking TATA boxes, where it can serve as an alternate staging site for polymerase II initiation. However, it is clear that the Inr sequence is by no means restricted to TATA-deficient promoters. Functional tests using the TATA-containing Drosophila gene Eip28/29 support the idea that the Inr is a facultative promoter element, required for efficient transcription under some conditions. For example, the Inr protects basal expression of Eip28/29 from the silencing effect of ecdysone response elements. In addition, the Inr is required for the function of an enhancer of basal activity in Eip28/29. We conclude that Inrs are promoter elements found sporadically throughout the higher eukaryotes, that the requirement for an Inr depends upon the array of other promoter elements which may be present in a given gene, and that Inrs may permit enhancers to discriminate among promoters.