To identify streptococcal genes that are expressed during experimental endocarditis, we developed a promoter-less dual reporter gene-fusion (amy, cat) plasmid, pAK36. Chromosomal DNA from S. gordonii V288 was digested with Sau3A1. The resulting fragments were ligated into pAK36. Following transformation into S. gordonii, the library of random gene fusion clones was inoculated into a rabbit to induce experimental endocarditis. Chloramphenicol treatment effected positive selection. Upon euthanization of the rabbits, the valvular vegetations were excised in a sterile field. Surviving clones were isolated and screened in vitro for chloramphenicol sensitivity and negative amylase activity. From the 48 randomly picked, double-negative clones, DNA was isolated and analyzed by Southern hybridization with labeled pAK36 probe. Different insertion patterns were identified, suggesting that no fewer than 13 S. gordonii genes were induced. Therefore, S. gordonii genes are induced during experimental endocarditis, which may contribute to virulence.