Previously, it was shown that inactivation of the tricarboxylic acid cycle aconitase gene acnA impairs the morphological and physiological differentiation of Streptomyces viridochromogenes Tü494, which produces the herbicide phosphinothricin tripeptide (PTT). In order to further characterize the role of the aconitase in the Streptomyces life cycle, aconitase activity was analyzed during growth of S. viridochromogenes in liquid culture. Two prominent maxima were measured in cell-free crude extracts. The first maximum was found at an early stage of growth, which is correlated with a decrease in pH when rapid glucose consumption is initiated. The second, lower maximum was detected at the beginning of the expression of the PTT-specific biosynthetic gene phsA, implying the onset of secondary metabolism. These results were confirmed by examining transcription of the acnA promoter in time-course experiments. The highest transcription rate was found during the early growth phases. In order to identify putative regulatory mechanisms, the transcriptional start site of the acnA transcript and subsequently the promoter were identified. Several putative, regulatory protein binding sites (e.g. regulators of oxygen stress or iron metabolism) were detected in the promoter region of acnA, which suggested complex regulation of acnA.