The identification of environmentally regulated genes of Streptococcus suis by the use of iron-restricted conditions in vitro and by experimental infection of piglets is described. Eighteen unique iron-restriction-induced (iri) genes and 22 unique in-vivo-selected (ivs) genes of Strep. suis were found. None of the ivs genes was exclusively expressed in vivo. Four iri genes were identical to four clones selected in piglets. Two ivs genes were similar to genes for putative virulence factors. One of these ivs genes was identical to the epf gene of virulent Strep. suis serotype 2 strains and the other showed homology to a gene encoding a fibronectin-binding protein of Streptococcus gordonii. Two additional ivs genes showed homology to environmentally regulated genes previously identified by using an in vivo expression technology (IVET) selection system in other bacterial species. One of these showed similarity to the agrA gene of Staphylococcus aureus, a key locus involved in the regulation of numerous virulence proteins. The promoter selection system described in this paper has been successfully used for the identification of many environmentally regulated genes potentially involved in the pathogenesis of Strep. suis infections in piglets.