To study streptococcal genes that are specifically induced in the host during endocarditis, we have developed a novel plasmid for use in in vivo expression technology (IVET). This IVET uses an integration plasmid, pAK36, that carries dual (amy-cat) reporter genes. A gene-fusion strain library was constructed with the plasmid randomly inserted into the chromosome of Streptococcus gordonii V288 by insertion-duplication. The library was inoculated intravenously into a rabbit that had been prepared for experimental endocarditis. Beginning 6 h after the inoculation, the rabbit was given chloramphenicol (Cm) intravenously twice a day to a final serum level of 5 μg/ml and was euthanized 3 days later. The aortic valve vegetations containing Cm(R) S. gordonii clones were cultured. Colonies were screened in vitro for negative amylase activity and sensitivity to Cm. Forty- eight such colonies showed 13 different insertion patterns when Southern hybridization blots were probed with labeled pAK36. For each of the 13 isolates, the gene fragment proximal to the insertion of the reporter amy-cat was cloned, and its nucleotide sequence was determined. Functions of these genes were inferred by their homology to known genes. Therefore, this novel IVET vector can be useful for identification of in vivo induced genes in S. gordonii and other streptococcal species.