Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5' tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055T. Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.