A novel LIM-homeodomain gene, Lhx9, was isolated by degenerate RT-PCR followed by mouse embryonic library screening. Lhx9 cDNA encodes a protein that is most closely related to Drosophila apterous and rodent Lhx2 proteins. The Lhx9 spatiotemporal pattern of expression during embryogenesis was similar but distinct from Lhx2. Highest expression levels were found in the diencephalon, telencephalic vesicles, and dorsal mesencephalon. Domains of expression respected the proposed neuromeric boundaries (). Lhx9 was also expressed in the spinal cord, forelimb and hindlimb mesenchyme, and urogenital system. Although Lhx9 expression was sustained in diencephalon and mesencephalon from embryonic day 10.5 (E10.5) to postnatal stages, it was transient in the future cerebral cortex, where it was turned off between E14.5 and E16.5. Lhx9 expression was highest if not exclusively located (depending on the region of interest) in the intermediate and mantle zones, as opposed to the mitotic ventricular zone. Lhx9 protein was tested for interaction with the recently discovered cofactors of LIM-homeodomain proteins and was found to interact strongly both with CLIM1 and CLIM2. The expression pattern and structural characteristics of Lhx9 suggest that it encodes a transcription factor that might be involved in the control of cell differentiation of several neural cell types. Furthermore, Lhx9 protein could act in a combinatorial manner with other LIM-homeodomain factors expressed in overlapping pattern.