Mouse embryonic mesenchymal cells undergo spontaneous smooth muscle (SM) differentiation upon spreading/elongation in culture (Relan et al., J. Cell Biol. 147 (1999) 1341; Yang et al., Development 125 (1998) 2621; Yang et al., Development 126 (1999) 3027). Using these cells we generated a subtracted cDNA library to identify potential suppressors of SM myogenesis. One of the differentially expressed genes was heterogeneous nuclear ribonucleoprotein-H (hnRNP-H), which is involved in pre-mRNA alternative splicing. hnRNP-H was highly expressed in mesenchymal cells prior to the onset of SM differentiation, but its expression rapidly decreased in mesenchymal cells undergoing SM myogenesis. In vivo, the drop in hnRNP-H expression was restricted to visceral SM cells. Antisense oligodeoxynucleotide and antisense RNA were used to inhibit hnRNP-H synthesis in SM-differentiating mesenchymal cells and in embryonic lung explants. A decrease in hnRNP-H levels resulted in upregulation of SM-specific gene expression and increased bronchial SM development in lung explants. hnRNP-H overexpression in cell cultures had the opposite effect. These studies, therefore, indicate a novel role for hnRNP-H in the control of visceral myogenesis.