Our aim was to improve our molecular understanding of the type 2 peritrophic matrix (PM) in Diptera. This was achieved by identifying novel chitin binding proteins (peritrophins) in Drosophila adult and larval PMs upon isolating cardia-specific cDNAs. A differential library screening strategy yielded ten cardia-specific clones. A (mostly Fluorescent) In Situ Hybridisation approach was used to accurately determine the site of peritrophin transcription in wholemount cardia. The images obtained provide evidence of clear-cut zones of peritrophin expression within the cardia epithelium, used to infer the particular PM layer into which these proteins are inserted, and thus predict their distribution within the mature PM. Attempts to set up PM-compromised fly lines in which specific peritrophins were knocked down using RNA interference were confounded by redundancy. The deficiency Df(3L)vin3, causing maximal disruption to larval PM integrity, served to test the protective function of the PM. Mortality was three-fold greater in deficient compared to wild-type larvae infected with pathogenic bacteria. Quantitative Reverse Transcription-PCR studies were performed to assess the modulation of peritrophin transcription in response to physiological fluctuations. Despite two days starvation, peritrophin transcription levels in older flies are three times higher than in newly eclosed flies. peritrophin transcription was upregulated following p er os bacterial infection in larvae only. In Glossina, eight putative peritrophins were identified with the aid of bioinformatics, five of which share similarities with known PM proteins. Transcripts were preferentially expressed in the cardia of G. morsitans morsitans, G. pallidipes and G. palpalis, although they were not modulated in response to trypanosome infection. The homologs showed little variation in the hypervariable regions between conserved cysteine and aromatic regions characteristic of chitin-binding domains. The results demonstrate the potential link between peritrophins and PM structure, and thus function. A number of remaining questions, worthy of future investigation, are suggested.