Subtractive suppressive hybridization was used to identify differentially expressed genes in subcuticular tissues from white spot syndrome virus(WSSV)-infected shrimp kept at different temperatures. Subtractive libraries I and II contained genes expressed at 26 and 33 degrees C, respectively. Three hundred and seventy-nine insert positive clones were selected to confirm differential expression by dot-blot hybridization. Twenty-two clones from library I and eight from library II were sequenced. All sequences from Library I corresponded to white spot syndrome virus genes. From library II, five clones were homologous with previously reported expressed sequence tags of Litopenaeus vannamei, two had similarity with beta-actin and one transcript represented an unknown gene. Over-expression of VP15 in shrimp at 26 degrees C was further confirmed by real-time polymerase chain reaction (PCR), whereas beta-actin expression was similar in animals kept at both temperatures. Together, our results show that hyperthermia reduces the expression of WSSV genes on shrimp subcuticular epithelial cells.