The polyembryonic parasitoid Copidosoma floridanum produces two larval castes, soldiers and reproductives, during development within its host. Soldier larvae defend the brood against competitors while reproductive larvae develop into adult wasps. As with other caste-forming insects, the distinct morphological and behavioral features of soldier and reproductive larvae likely involve differential gene expression. In this study we used a bi-directional suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes from C. floridanum soldier and reproductive larvae. We isolated 230 novel expressed sequence tags (ESTs) from the two subtractions (114 soldier/116 reproductive ESTs). Among these ESTs were sequences with significant similarity to genes coding for serine proteinases, proteinase inhibitors, odorant-binding and chemosensory proteins, and cuticular proteins. Also, three novel genes were isolated that resemble one another in conceptual translation and share the cysteine spacing pattern of short scorpion toxins and insect defensins. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of 20 ESTs from the two libraries indicated that 85% were differentially expressed in one caste or the other. We conclude that our SSH strategy was effective in identifying a number of genes differentially expressed in soldier and reproductive larvae and that several of these genes will be useful in characterizing caste-specific gene networks in C. floridanum.