A method for obtaining primary cultures of epithelial cells from rainbow trout gills is described. The yield of cells from approximately 1.5 g wet mass of tissue was 218×106+/−12×106 cells with a viability defined by eosin exclusion of 80+/−6 %. Cells were seeded in culture dishes and grown in Leibowitz L-15 medium supplemented with 5 % foetal bovine serum. Attachment efficiency after 24 h was 35+/−6 %. The cells appeared confluent 10–12 days after seeding and exhibited surface structures similar to those seen on respiratory epithelial cells of trout gills in vivo. Growth rate, [3H]thymidine incorporation and attachment efficiency were used to evaluate culture conditions. Epidermal growth factor, insulin, transferrin, hydrocortisone, laminin and collagen did not improve growth and attachment. Similarly, coating the culture dishes with rat tail collagen, trout skin extract, laminin or a mixture of human basement membrane proteins (Matrigel) failed to improve attachment. It is concluded that the cells in culture are respiratory epithelial cells and that this culture system could provide a valuable new approach for studying the physiology of these cells.

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