Escherichia coli Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes. A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control. However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant. In this work, 72 independent mutations were generated in the fis promoter (fis P) region from -108 to +78 using both random and site-directed PCR mutagenesis. beta-Galactosidase activities from mutant promoters fused to the (trp-lac)W200 fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the -35 and -10 regions, respectively, which are optimally separated by 17 bp. We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the -35 region, but not in the -10 region, significantly reduced the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of fis P mRNA were detected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the -10 and -35 consensus sequences. These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the -10 and -35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.