Establishment of receptive endometrium is essential for implantation. Our aim was to identify and characterize genes uniquely regulated at the sites of implantation in mouse uterus by RNA differential display polymerase chain reaction (DDPCR). One of the gene fragments identified was 86% homologous to rat calcium-binding protein D9k (calbindin-D_9k); the mouse counterpart had not then been cloned, but subsequently an mRNA sequence of mouse calbindin-D_9k became available in GenBank (accession number: AF028071). This sequence is 99% homologous to the DDPCR-derived gene tag but has a shorter 3′ end. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the sequence of 3′ end of the DDPCR product and the 5′ end of AF028071, and a full cDNA was obtained. This gene was primarily up-regulated by progesterone, but not by estrogen. It was further increased by the combination of the two steroids. Expression of calbindin-D_9k was overall increased in the uterus during early pregnancy, but the level was significantly lower in implantation compared to interimplantation sites on Days 4.5 and 5.5 of pregnancy, becoming barely detectable in both sites after Day 6.5. In situ hybridization localized this mRNA predominantly in the luminal epithelium of the pregnant uterus. The complex regulation of calbindin-D_9k in mouse uterus suggests an important role for this protein during pregnancy.