To investigate functions of the homeodomain-containing transcription factor Nkx3.1 a null mutation was generated by targeted gene disruption introducing the bacterial LacZ gene as reporter into the locus. In addition to defects in duct morphogenesis of the prostate and bulbourethral gland displaying progressive epithelial hyperplasia and reduced ductal branching (Bhatia-Gaur, R., Donjacour, A.A., Sciavolino, P.J., Kim, M., Desai, N., Young, P., Norton, C.R., Gridley, T., Cardiff, R.D., Cunha, G.R., Abate-Shen, C., Shen, M.M., 1999. Genes Dev. 13, 966-977), we observed a novel phenotype in minor salivary glands of Nkx3.1 null mutants. Minor salivary glands in the oral cavity of mutant mice appeared reduced in size and exhibited severely altered duct morphology. Other Nkx3.1 expressing regions were unaffected by the mutation. The activity of the Nkx3. 1/LacZ allele faithfully reflected the known expression domains of Nkx3.1 in sclerotome, a subset of blood vessels, Rathke's pouch, and ductal epithelium in prostate and minor salivary glands during pre- and postnatal mouse development. However, it was additionally expressed in the heart, duodenum and lung. These ectopic expression domains resemble the pattern of the Nkx2.6 gene which is closely linked to Nkx3.1 in the mouse genome and its regulation may therefore be affected by the mutation. In Nkx3.1/Shh compound mutant mice we found that Nkx3.1 expression in sclerotome and prostate was strictly dependent on sonic hedgehog (Shh) signaling, while other expression domains including heart and gut were independent of Shh. Expression in lung appeared augmented in the absence of Shh. Our results suggest that Nkx3.1 plays a unique role in regulating proliferation of glandular epithelium and in the formation of ducts in prostate and minor salivary glands.