The cDNAs encoding antimicrobial peptides (AMPs) in the skin of Hylarana guentheri were identified,namely temporin (five peptides,termed temporin-GHa-GHd and temporin-GUa),brevinin-1 (one peptide,brevinin-1GUb),and brevinin-2 (eight peptides,brevinin-2GHd-2GHj,and brevinin-2GHb).Eleven of the 14 peptides have novel primary structures.Synthesized temporin GHa-GHd have broad-spectrum antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis),Gram-negative bacteria (Escherichia coli,Vibrio alginolyticus,and Pseudomonas aeruginosa),as well as fungus (Candida a/bicans).Among these tested strains,S.aureus was the most sensitive to temporin-GHa-GHd with minimum inhibitory concentration (MIC) values between 6.8 and 12.9 μM.They also exhibited antimicrobial activities against Methicillin-resistant S.aureus with the MIC ranging from 12.7 to 51.7μM.Interestingly,secondary structure prediction shows that there is no α-helix in temporin-GHb,which illustrates that α-helix is not required for the antimicrobial activity of temporin-GHb.NaCl (at final concentrations of 0.15-2 M) decreased the antimicrobial activity of temporin-GHa-GHd slightly,while human serum and S.aureus V8 proteinase had no effect on the antimicrobial activity.Scanning electron microscopy images of E.coli and S.aureus showed that the surface of microbial cells was considerably rough and shrived after 1 h of treatment with temporin-GHa-GHd at 37℃.The stabilities of temporin-GHa-GHd in human serum or in S.aureus V8 proteinase make them to be promising candidates of novel antimicrobial agents or models for the development of novel AMPs.