Lhx9 is a LIM-homeodomain (HD) transcription factor. Transcripts of the Lhx9 gene are present in the urogenital ridges of mouse embryos. In Lhx9-deficient mice, a discrete gonad is not formed (Birk et al., 2000). To date, however, the role of Lhx9 in gonad formation has not been studied in animals other than chicks and mice. Thus, this study was aimed at determining whether or not Lhx9 is expressed in the developing gonads of amphibians. We first isolated cDNAs of Lhx9 and four isoforms, named Lhx9α, β, γ and δ, from the frog Rana rugosa. All the isoforms lacked the last 14 amino acids of the HD (the DNA-binding motif). RT-PCR analysis revealed that Lhx9 and Lhx9α were highly expressed in the ovary, testis, brain and heart of adult frogs. However, neither Lhx9 nor any of the four isoforms was expressed in the pancreas and kidney. When their expression in the developing gonads of R. rugosa was examined by RT-PCR, transcripts of Lhx9 and Lhx9α were detected with no sexual dimorphism during sex determination. Lhx9γ and Lhx9δ were also expressed, but at a low level. In contrast, the Lhx9β transcript was hardly detected. In addition, in situ hybridization analysis showed that R. rugosa Lhx9 was expressed in somatic cells in the developing gonad and mesonephros far before sex determination. Taken together, Lhx9 and its four isoforms are probably involved in organogenesis in R. rugosa. All the isoforms may function as an endogenous dominant-negative form of Lhx9 in this species.