Epithelial cells represent one of the most important physical barriers to many bacterial pathogens. In the case of Neisseria gonorrhoeae, the epithelial cell response is critical because they are the main target of the tissue damage triggered by the pathogen, particularly when the organism reaches the Fallopian tube (FT). Although the irreversible damage triggered by N. gonorrhoeae in the FT has been previously reported (ectopic pregnancy, pelvic inflammatory disease and infertility), the mechanisms of gonococcal-induced tissue damage are not fully understood. In addition, the lack of animal models that efficiently mimic the human disease and the complexity of gonococcus–host interactions make studying gonococcal pathogenesis particularly difficult. The use of human immortalized cells is also limited, since a variety of commercial FT cell lines is not yet available. Finally, the phase and antigenic variation of many gonococcal surface molecules involved in attachment and invasion of epithelial tissues leads to a failure to reproduce results using different human cells lines used in previous studies. The FT organ in culture (FTOC) and primary human fallopian tube epithelial cell (FTEC) represent the closest ex vivo cell models to explore the biology of Neisseria gonorrhoeae during infection of the FT, since it is a natural host target of the gonococcus. In this chapter, we describe protocols to process human FT samples to obtain FTOC and FTEC and assess their response to infection with Neisseria gonorrhoeae.