Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.