Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon. A total of 1062 expressed sequence tag (EST) clones were sequenced unidirectionally. ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered. They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively. Several types of each AMP homologue were found. Sequence alignments of ALF in P. monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini. Only one major type of penaeidin (penPm3) was found in P. monodon. In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized. Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes. Expression of these antimicrobial peptides in P. monodon subjected to V. harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins.