Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.