Escherichia coli D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is produced by the gapA gene and is structurally related to eukaryotic GAPDHs. These facts led to the proposal that the gapA gene originated by a horizontal transfer of genetic information. The yields and start sites of gapA mRNAs produced in various fermentation conditions and genetic contexts were analyzed by primer extension. The transcriptional regulatory region of the gapA gene was found to contain four promoter sequences, three recognized by the vegetative RNA polymerase Eff70 and one recognized by the heat shock RNA polymerase E&32. Transcription ofgapA by Eu32 is activated in the logarithmic phase under conditions of starvation and of heat shock Using a GAPDH-strain, we found that GAPDH production has a positive effect on cell growth at 43°C. Thus, E. coli GAPDH displays some features of heat shock proteins. One of the gapA promoter sequences transcribed by Eu70 is subject to catabolic repression. Another one has growth phase-dependent efficiency. This complex area of differentially regulated promoters allows the production of large amounts of gapA transcripts in a wide variety of environmental conditions. On the basis of these data, the present view of Eu32 RNA polymerase function has to be enlarged, and the various hypotheses on E. coli gapA gene origin have to be reexamined.