The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kμ cm²) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na fluxes and application of the Ussing flux ratio criterion demonstrated active Na uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca² uptake and Na /K -ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.