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Background  Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence  in situ  hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful  in silico  design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.    Results  The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and  in situ  target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool.    Conclusion  Using this tool,  in silico  probe design and evaluation can be performed with respect to  in situ  probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of  in situ  hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page   .

Graphical representation of ribosomal RNA probe accessibility data using ARB software package.