We have previously cloned and sequenced the ppa gene, encoding inorganic pyrophosphatase (PPase), of Escherichia coli K12 [Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) Journal of Bacteriology 170, 5901-5907]. In this work mutations were constructed in the 5' flanking region of E. coli ppa and the effect on expression was determined. The minimum length of the fully active ppa5' flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide -37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide -50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the -50 to +20 region in E. coli genes. When the -35 sequence of ppa, AAGACA, was mutated to AAAACA, ppa expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the -10 sequence, TATAAT, to TTTAAT or TATAAA, the ppa gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically. The implications of these results are discussed.