Microarrays are powerful tools for analyzing and understanding genome functionality. To run microarray experiments, one needs to calibrate many consecutive steps in order to avoid variability and to allow experiment comparisons. In this work, we have developed ROSO (“logiciel de Recherche et Optimisation de Sondes Oligonucléotidiques”), a software devoted to design optimal oligonucleotide long probes (> 30 bp) for microarrays. ROSO allows users to choose the type of probes, their size and localization on the gene target, the number of probes per gene target, as well as different hybridization parameters such as ion concentrations, melting temperature range and threshold for secondary structure rejection. ROSO is a web tool accessible at the following URL (http://pbil.univ-lyon1.fr/roso/loadWkgFiles.php). Probe optimization process is based on four successive key steps: 1.Probe specificity. Specificity is calculated by comparing probes to the overall studied set of genes (internal genes), but also by comparison with an external set of genes (any genes user want to avoid any cross-hybridization with). Specificity is calculated with Blast program. Blast parameters were estimated to detect a minimal homology of 70 % on 20 nucleotides length. 2.Probe secondary structures (hairpin and homoduplex). Stability of secondary structures is calculated with the thermodynamic model of nearest-neighbor. 3.Probe melting temperature (Tm). Tm is calculated with the thermodynamic model of nearest-neighbor. When at least one probe is found for each gene, ROSO keeps the set of optimal probes with the smallest possible Tm variability. 4.Stabilizing criteria. When multiple probes are found for one target gene, stability criteria (GC rate, GC clamp…) are calculated and allows to fiind the best probe. Moreover, it allows users to calculate Tm of control probes with mismatches. Different kinds of validation were performed. First, simulated data have allowed for the comparison with the reference software Oligo6® and Mfold. ROSO estimations of Tm and secondary structures were found to be equivalent or better than Oligo6® and Mfold estimations, for probes size comprised between 15 and 70 nucleotides. Second, ROSO was used to design two sets of 541 and 609 probes for specific bacterial microarrays corresponding to Buchnera aphidicola and Ralstonia solanacearum. Human and murine probe sets were also designed. The work is conducted in collaboration with UMR 5558 (UCB Lyon) and with support of the Genopôle Rhône-Alpes.