The antimicrobial peptides (AMPs) are short, highly positively-charged peptides of 20-40 amino acids in length with broad spectrum antimicrobial activity, believed to be important components of epithelial defence (Lehrer & Ganz 2002). The aim of the present study was to compare AMP expression and localisation between human palatine tonsils and HEp-2 cells from an epidermoid cancer of the larynx, an in vitro human epithelial model widely used to study tonsillitis (Eyal et al. 2003). Palatine tonsils were harvested with ethical approval and after informed consent from 19 patients with recurrent acute tonsillitis (during disease-free phase) and 5 control subjects undergoing tonsillectomy for sleep disorders. The surface epithelium was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated and AMP expression characterised using reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for AMPs, and compared with results from the Hep-2 cell line. Fluorescent immunohistochemical techniques were used to localise AMPs within fresh frozen tonsil sections, imaged with a Leica TCS-NT laser scanning confocal microscope. Products of the predicted size were generated by RT-PCR for each AMP and identity (100%) was confirmed by nucleotide sequence. We confirmed human palatine tonsils as a site of human β-defensin 1, 2 and 3 and LEAP-1 expression (Chae et al. 2001; Harder et al. 2001). We also demonstrated expression of two additional antimicrobial peptides LL-37 (cathelicidin I) and LEAP-2. All six AMPs were expressed in all 26 tonsil samples. Human β-defensins 1-3 and LL-37 showed similar localisation by immunohistochemistry to the oropharyngeal surface and crypt epithelia. In contrast, the in vitro HEp-2 cell model expressed only human β-defensin 1 and 3. Induction of the four ‘missing’ AMPs in Hep-2 cells was not observed after a three hour challenge with 10⁷ CFU/ml of M1 wild-type Group A Streptococcus. A range of AMPs are expressed in the human tonsillar epithelium, suggesting a role in protecting the tonsil epithelium from infections. Hep-2 cells do not express the same range of AMPs as tonsils, questioning use as a model in the study of tonsillitis.