Hox genes code for transcription factors that define pattern formation and organ structure in the developing embryo. We have previously shown that the expression pattern and antisense inhibition of Hoxb-5 in the developing mouse lung suggests its importance to proximal airway branching and epithelial cell fate. We hypothesized that stimulation of Hoxb-5 in embryonic lung would alter the normal pattern of branching morphogenesis. We used retinoic acid (RA) to induce Hoxb-5, and Hoxb-5 antisense or sense oligonucleotides (Hoxb-5 AS or Hoxb-5 sense) to determine if the changes in branching morphogenesis related to RA treatment are Hoxb-5 specific. Embryonic mouse lungs (d11.5, term is 19 days) were cultured in BGJb medium with ethanol (control); RA (10-6M) alone; RA in combination with Hoxb-5 AS (20 μM) or Hoxb-5 sense (20 μM) oligos; or with Hoxb-5 AS (20 μM) or Hoxb-5 sense (20 μM) oligos alone for 72 hrs. Changes in morphology and terminal bud counts were evaluated at 24 hr intervals in control versus treatment groups. At 72 hrs, lungs were harvested and Hoxb-5 protein analyzed by Western blot and immunostaining. As compared to ethanol and sense controls, RA treated lungs had elongated proximal branches with decreased distal branching. In embryonic lungs treated with RA and Hoxb-5 AS oligos, the morphologic changes seen with RA treatment alone were prevented and in contrast showed decreased elongation of proximal branches and decreased distal cleft development leading to foreshortened dilated secondary branches with multiple rudimentary distal clefts. Embryonic lungs treated with Hoxb-5 AS oligos alone showed similar findings to lungs treated with RA in combination with Hoxb-5 AS oligos. As compared to ethanol and sense treated cultures, terminal bud counts were reduced by 25%(p<0.02)in lungs treated with RA or with RA/Hoxb-5 AS oligos. Western blot analysis showed a 200-300% increase in Hoxb-5 protein in RA versus control lungs. Conversely, Hoxb-5 protein was decreased in RA/Hoxb-5 AS and Hoxb-5 AS treated lungs as compared to control, sense and RA treated lungs. Histochemistry of RA treated lungs showed elongated, dilated proximal airways lined by cuboidal epithelium. Hoxb-5 immunostaining was increased in the subepithelial mesenchyme adjacent to these dilated, elongated airways. We conclude that regional and quantitative changes in Hoxb-5 protein expression alters branching morphogenesis of proximal airways in the developing mouse lung. Alterations in branching morphogenesis induced by RA treatment are mediated in part through Hoxb-5 stimulation.