Surfactant-associated proteins (SP) play an important role in the function of pulmonary surfactant. We have previously shown that SP-B mRNA is increased whereas SP-A and SP-C mRNA are decreased by all-trans-retinoic acid (RA) in a dose-dependent manner in human fetal lung explants. All-trans-RA binds primarily to the retinoic acid receptors (RARs) and 9-cis-RA binds primarily to the retinoid X receptors (RXRs). Because the fetal lung contains RXRs, we hypothesized that 9-cis-RA regulates surfactant protein gene expression in lung epithelial cells. H441 human lung adenocarcinoma cells, which synthesize SP-A and SP B mRNA and protein, were treated with either all-trans-RA or 9-cis-RA (10-10 to 10-6 M) for 24 h. Neither all-trans- RA nor 9-cis-RA had an effect on SP-A mRNA levels in the H441 cells. All- trans-RA (10-6 M) significantly increased SP-B mRNA levels in the H441 cells and 9-cis-RA had a smaller, not statistically significant effect. Human fetal lung explants were treated with 9-cis-RA for 6 d. 9-cis-RA did not significantly increase SP-B mRNA levels, significantly inhibited SP-A mRNA levels at all concentrations tested, and significantly inhibited SP-C mRNA levels at 10-6 M in the human fetal lung explants. Both all-trans-RA (10- 6 M) and 9-cis-RA (10-6 M) significantly increased SP B protein levels in the human fetal lung explants. Together, these results are suggestive that all trans-RA directly regulates SP-B gene expression in human pulmonary epithelial cells. In addition, the inhibitory effect of all-trans-RA and 9- cis-RA on SPA mRNA levels in pulmonary epithelial cells is probably an indirect effect mediated by other cell types present in fetal lung tissue.