The major digestive enzyme of Anopheles gambiae is encoded by the trypsin 1 gene. This gene is expressed exclusively in the gut and its mRNA abundance increases after ingestion of a blood meal. Previous experiments with transgenic Drosophila have shown that the enhancer region, from nucleotide -360 bp to -150 bp upstream of the transcription initiation site, is necessary to drive the gut-specific expression of a reporter gene (Skavdis et al., 1996. EMBO J. 15, 344-350). In this study, we defined DNA sequences within this region that are capable of binding nuclear factors from either gut or non-gut tissues. By electrophoretic mobility shift assays, we determined that a gut-specific nuclear factor recognizes and binds to three sites in the enhancer region with a consensus sequence TYCAAGT. Another factor, found in many tissues, recognizes and binds to at least two additional sites with a consensus sequence ACGATA. This study defines for the first time for an insect gut-specific enhancer, specific sequences that interact with nuclear factors.