To identify genes that are differentially expressed in the developing testis we used representational difference analysis of complementary DNA from gonads of mouse embryos at 13.5 days postcoitum (dpc). Three genes were identified. One of them was a novel gene termed tescalcin that encoded a putative EF-hand Ca(2+)-binding protein. The open reading frame consisted of 642 nucleotides encoding a protein with 214 amino acids. Analysis of the predicted amino acid sequence revealed an N:-myristoylation motif and several phosphorylation sites in addition to an EF-hand Ca(2+)-binding domain. TESCALCIN: messenger RNA (mRNA) was present in fetal testis, but not in ovary or mesonephros, and was restricted to the testicular cords. Its expression was first detected in the male gonad at 11.5 dpc and demonstrated a pattern consistent with a role in the testis at the early stages of testis differentiation. Tescalcin is expressed in the testis of Kit(W/W-v) mice, indicating that it is not dependent on the presence of germ cells. The other two genes identified were collagen IX alpha3 (Col9a3) and RENIN: Col9a3 expression was present at low levels in male and female gonads at 11.5 dpc. Thereafter, it was markedly up-regulated in the male, but remained very low in the female. Expression of Col9a3 was restricted to testicular cords and was also detected in testis of Kit(W/W-v) mice. RENIN: mRNA was first detected in testis at 12.5 dpc, increased thereafter, and reached a peak at 16.5 dpc. RENIN: mRNA was localized in cells of the interstitium and cells at the border between the gonad and mesonephros. Expression of RENIN: in the ovary was not detected using standard conditions.