A comprehensive culture-independent assay, called Q-FAST, was developed for concurrent identification and quantification of active microorganisms involved a specific function in a given microbial community. The development of Q-FAST was achieved by integrating the concept of stable isotope probing technique into a new quantitative fingerprinting assay called real-time-t-RFLP for microbial community structure analysis. The Q-FAST was successfully validated by using a three-member artificial microbial community containing a known naphthalene-utilizing bacterium (Pseudomonas putida G7) and two nonnaphthalene-degrading bacteria (Escherichia coli and Bacillus thuringiensis). The application of Q-FAST to identify and quantify a guild of naphthalene-utilizing microorganisms in soils revealed the involvement of eight members, with six members relating to several phylogenetic groups of eubacteria (three in beta-proteobacteria, two in gamma-proteobacteria, and one in genera Intrasporangium of Gram-positive bacteria) and two members showing no close phylogenetic affiliation to any known bacterial sequences deposited in GenBank. The quantity of three members belonging to beta-proteobacteria accounted for 34% of total 16S rDNA copies measured from the "heavier" fraction of DNA that was contributed from the DNA of microorganisms capable of incorporating 13C-labeled naphthalene into their genetic biomarkers. The other five members composed 66% of total 16S rDNA copies of active naphthalene-utilizing populations measured. Offering a powerful tool for studying microbial ecology, Q-FAST thus opens a new avenue for deeper exploration of microbial-mediated processes, mainly the quantitative relationship between microbial diversity and microbial activity in a given environment.