We have analyzed the relative activities of the Escherichia coli ribosomal RNA promoters P1 and P2 in vivo under different physiological conditions. Promoter efficiencies were determined by quantitative comparison of the transcript-specific primer extension products obtained from total RNA preparations. Cells were analyzed at different stages of the growth cycle, at different growth rates, and under conditions of stringent control. In addition, the rRNA gene dosage was altered by transformation with plasmids containing additional rrnD or rrnB transcription units, or rRNA operons in which one of the tandem promoters (P1) had been deleted. Under conditions of amino acid starvation (stringent control) we observed the expected strong reduction in P1-directed transcription. In contrast to the previous assumption that the P2 promoter is not regulated, we simultaneously noticed a smaller but significant repression of P2-directed transcription. In strains in which the rRNA gene dosage was increased by transformation with plasmids bearing rRNA transcription units, a similar degree of repression was observed. Repression of the P1 promoter activity was increased, however, when cells contained extra rRNA operons with P2 promoters only. As demonstrated under stringent control conditions, changes in the growth cycle also affected the activity of promoters P1 and P2. A greater proportion of P2-derived transcripts was observed when cells changed from exponential to stationary growth or if cultures were grown in minimal medium. Under steady-state, slow growth conditions (minimal medium) we obtained evidence showing that the ratio of P1/P2 transcription products is much lower for cells with extra rrnB as compared to extra rrnD operons or cells lacking extra rRNA operons, implying an operon-specific regulation.(ABSTRACT TRUNCATED AT 250 WORDS)