We reported elsewhere that mutation in the pgsA gene, responsible for the synthesis of phosphatidylglycerol, repressed the synthesis of flagellin and caused the loss of motility of Escherichia coli (Tomura et al., FEBS Letters 329, 287-290, 1993). We now describe evidence for a decrease in promoter activity of the flhD gene, a master gene for flagellum synthesis, in the pgsA3 mutant. We constructed a plasmid with a promoter region of the flhD gene connected with the structure region of the lacZ gene. The activity of beta-galactosidase in the extract prepared from the pgsA3 mutant harboring the fusion plasmid was 30% of that in the wild type cells. This result means that phosphatidylglycerol is likely to be required for the initiation of transcription of the flhD gene. We also found that the motility-less phenotype of the mutant was partially suppressed by elevating incubation temperature. This suppression is caused by restoration of transcription of the flhD gene by high temperature. As the content of phosphatidylglycerol did not increase by elevating incubation temperature, we proposed that this suppression is caused by alternation of a physical structure of phospholipid bilayers in cytoplasmic membranes.