Abstract: Aeromonas hydrophila is one of the main causative agents resulting in serious infectious diseases of turtles and other animals. To understand anti-infectious response to bacteria in reptile, a subtractlve cDNA library was constructed from the liver, spleen and kidney of Chinese soft-shelled turtle (Trionyx sinensis ) experimentally infected with A. hydrophila T4, using suppression subtractive hybridization (SSH). Experimental turtles were injected intraperitoneally with 1.6 ×10^8 CFU live A. hydrophila T4 and the control turtles were injected with steriled normal saline. The liver, spleen and kidney samples of infectious and control turtles were dissected out immediately, and frozen in liquid nitrogen for isolation of total RNA. About 500 mg tissues containing approximately equal amount of liver, spleen and kidney from infectious and control turtles were used as tester and driver samples, respectively. Total RNA extraction was performed from the two samples, followed by mRNA isolation. Using equal amounts of mRNA (2μg) from tester and driver samples, double-strand cDNA was synthesized and digested with restriction enzyme RsaI for three hours. The RsaI-digested tester was subdivided into two pools, and each was ligated with a different adaptor. The RsaI-digested driver cDNA was not exposed to adaptors. Then an excess of driver cDNA was added to each tester cDNA for the first round of hybridization to enrich for differentially expressed sequences. In the second, the two samples from the first hybridization were mixed together and freshly denatured driver DNA was added to further enrich for differentially expressed sequences. New molecules were formed which consist of differentially expressed cDNAs with different adaptors on each end. Then two rounds of suppression PCR were performed. In the first amplification, only ds cDNAs with different adaptor sequences on each end were selected and exponentially amplified. In the second, nested PCR was used to further reduce background and enrich for differentially expressed sequences. Turtle β-actin gene was used as internal control to estimate the efficiency of subtractive cDNA. In this library, β-actin was subtracted significantly at about 2^10 folds, suggesting that the subtractive cDNA library was successfully constructed. The PCR products were inserted into pMD18-T vector and transformed to competent E. coli DHSa cells to set up a subtracted and normalized PCR fragment library. PCR analysis showed that the inserts were 150-800bp in length. To our knowledge, this is the first report of a subtractive cDNA library constructed from experimentally bacteria infected tissue in reptile. A serials of immune-relevant genes, such as IL-8, CD9, CD59, SAA, and ISG12 were first isolated and cloned in turtles (will report in another paper), and the IL-8, CD9, CD59 and ISG12 genes were also first isolated in reptile. The successfully constructed cDNA library will be essential for rapid isolation of differentially expressed genes related to A. hydrophila infection, and useful for understanding the anti-infectious molecular mechanism in reptile.