A simple method for the quantification of uncultured microorganisms in the environment was developed. In vitro-transcribed 16S rRNA is used as a template for midpoint dissociation temperature (Td) determinations of specific oligonucleotide probes and as a standard in quantitative probing. It replaces the need for total nucleic acids extracted from pure cultures of the organisms to be quantified. A sense RNA of a size almost identical to that of native 16S rRNA can be transcribed from ribosomal DNA clones recovered in studies of the phylogenetic diversity of microbial communities. This in vitro-transcribed rRNA yields dissociation curves typical of oligonucleotides. They parallel curves determined with total nucleic acids but yield slightly higher Td values. Neither unspecific sticking of the probe nor probe washing off the DNA template at low temperatures fully accounted for the discrepancy in probe release from the two templates. This suggests that the native rRNA itself has melting characteristics different from those of its in vitro-transcribed counterpart. However, this difference does not affect the performance of in vitro-transcribed rRNA compared with total nucleic acids as a standard in quantitative hybridizations. No difference was found between the estimates of the relative quantity of a single bacterial species in a mixed community obtained with the two standards, regardless of whether DNA was removed from the samples. This protocol will allow the large-scale quantification of the ecological importance of uncultured microorganisms in natural environments for the first time.