HOM-C/Hox genes are important regulatory genes that control structural polarity, cell fate, and pattern formation in vertebrate development. Retinoic acid (RA) activates and regulates these Hox genes in a dose- and time-dependent as well as cell-specific manner. Hoxa-5 is highly expressed in the fetal mouse lung but its exact function in lung development is unknown. We hypothesized that RA regulates Hoxa-5 gene expression in fetal mouse lung in a cell-specific fashion. Primary cultures of E17.5 fetal mouse lung fibroblasts, and MLE 12 cells (transformed mouse lung tumor cells that have characteristics of alveolar type II cells) were grown to confluence in separate tissue culture dishes. Cells were treated with RA (10-8 M to 10-4 M) for 3, 6, 14, 24, and 48 hours. Hoxa-5 mRNA was analyzed in Northern blots. Fibroblasts showed a dose-dependent stimulation of Hoxa-5 gene expression by RA. Peak stimulation occurred within 3 to 6 hours of treatment for every RA concentration used. Subsequently, with increasing exposure time to RA, expression decreased with the lower doses of RA (10-8 M to 10-6 M) but not with the higher doses (10-5 M to 10-4 M). In contrast, maximal Hoxa-5 gene stimulation in MLE 12 cells occurred later at 14 hours, then decreased to baseline by 48 hours. Untreated MLE 12 cells also expressed Hoxa-5 mRNA. For both cell types, the degree of gene induction increased with increasing doses of RA. We conclude that RA regulates cell-specific Hoxa-5 gene expression in fetal lung fibroblasts and MLE 12 cells in a dose- and time-dependent manner. Maximal stimulation of Hoxa-5 expression occurs 8 to 11 hours earlier in fibroblasts compared to MLE 12 cells, suggesting a process of mesenchymal-epithelial interaction in which the delayed stimulation of Hoxa-5 in the epithelial cells functions to sustain processes previously started earlier in the mesenchymal fibroblasts. The differences in Hoxa-5 gene expression between the two cell types is a function of unknown RA-mediated factors. The expression and activation of Hoxa-5 in the lung are not restricted to fibroblasts but occur in epithelial cells as well. This ability of RA to activate Hoxa-5 in MLE 12 epithelial cells indicates a potential role of Hoxa-5 in regulating differentiation of pulmonary epithelium. We speculate that Hoxa-5 plays an important role in mouse lung development, possibly in pulmonary epithelial differentiation.