The goal of our studies is to elucidate mechanisms that control and modulate mucous differentiation and mucin gene expression in the conducting airways. We used cultures of normal human tracheobronchial epithelial (NHTBE) cells that were shown to secrete two major airway mucins, namely MUC5AC and MUC5B as well as several other secretory products. Mucous differentiation and expression of MUC2, MUC5AC, MUC5B and MUC7, but not MUCi, MUC4, and MUC8 mucin genes, were shown to be retinoic acid- (RA) or retinol-dependent. We found that RA control of mucin genes was mediated by the retinoid acid receptors RAR alpha and, to a lesser extent, by RAR gamma. Our studies also showed that other important bioregulators such as thyroid hormone (T3) and epidermal growth factor (EGF) modulate basal expression of mucin genes, interacting with RA in a concentration-dependent manner. T3, which binds to thyroid receptors (TRs) belonging to the same superfamily of steroid hormone nuclear receptors as the RARs, inhibits mucin gene expression, particularly MUC5AC. One possible mechanism of this T3 effect is downregulation of RAR proteins, which are critical for mucin gene expression. However, we also found that T3 inhibits MUC5AC transcription.EGF, which had previously been shown to stimulate mucin expression and mucin secretion in cultured rat tracheal epithelial (RTE) cells, inhibited mucin secretion in human bronchial epithelial cell cultures. This effect was EGF concentration- and time-dependent and was progressively abolished by increasing the RA concentration. Subsequent studies suggested that the inhibitory effects of high concentrations of EGF may result from selective reduction of MUC5AC expression. These studies thus point to potentially important species differences in the mechanisms regulating mucous production, and they also confirm previous findings indicating differential regulation of MUC5AC and MUC5B gene expression.