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The purpose of this study was to subculture normal human nasal epithelial (NHNE) cells without compromising their ability to differentiate into secretory and ciliated cells and to study the effect of retinoic acid on mucous and serous secretions in passaged cells and to compare the expression of mucin and lysozyme in cultured cells with those in in vivo nasal epithelium. The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface cultures. The cultured NHNE cells secreted mucin and lysozyme. The cells became squamous and mucin secretion decreased when retinoic acid was deleted from the culture media. Cells from passage 1 through passage 2 remained able to differentiate into mucous or squamous cells. Mucin gene 4 (MUC4), MUC5AC, MUC7, MUC8, and lysozyme messenger RNAs were expressed in passage 2 NHNE cells. In conclusion, passage 2 NHNE cell cultures retain features of normal epithelium and are suitable for many studies of upper airway cell biology.