Leishmaniasis is an important worldwide public health problem. Visceral leishmaniasis caused by Leishmania infantum chagasi is mainly transmitted by Lutzomyia longipalpis in the Americas. Leishmania development within the sand fly vector is mostly restricted to the midgut. Thus, a comparative analysis of blood-fed versus infected midguts may provide an invaluable insight into various aspects of sand fly immunity, physiology of blood digestion, and, more importantly, of Leishmania development. To that end, we have engaged in a study to identify expressed sequenced tags (ESTs) from L. longipalpis cDNA libraries produced from midguts dissected at different times post blood meal and also after artificial infection with L. i. chagasi. A total of 2,520 ESTs were obtained and, according to the quality of the sequencing data obtained, assembled into 378 clusters and 1,526 individual sequences or singletons totalizing 1,904 sequences. Several sequences associated with defense, apoptosis, RNAi, and digestion processes were annotated. The data presented here increases current knowledge on the New World sand fly transcriptome, contributing to the understanding of various aspects of the molecular physiology of L. longipalpis, and mechanisms underlying the relationship of this sand fly species with L. i. chagasi.