The acceptance of rRNA sequence diversity as a criterion for phylogenetic discrimination heralds the transition from microbiological identification methods based on phenotypic markers to assays employing molecular techniques. Robust amplification assays and sensitive direct detection methods are rapidly becoming the standard protocols of microbiology laboratories. The emergence of peptide nucleic acid (PNA) from its status as an academic curiosity to that of a promising and powerful molecular tool, coincides with, and complements, the transition to rapid molecular tests. The unique properties of PNA enable the development of assay formats, which go above and beyond the possibilities of DNA probes. PNA probes targeting specific rRNA sequences of yeast and bacteria with clinical, environmental, and industrial value have recently been developed and applied to a variety of rapid assay formats. Some simply incorporate the sensitivity and specificity of PNA probes into traditional methods, such as membrane filtration and microscopic analysis; others involve recent techniques such as real-time and end-point analysis of amplification reactions.