We have identified and characterized a cDNA encoding a novel FK506-binding protein (FKBP), named FKBP23, from mouse heart by the signal sequence trap method. The deduced amino acid sequence has significant homology to other FKBP family members around the peptidylprolyl cis-trans-isomerase motifs. FKBP23 also has two Ca2+-binding (EF-hand) motifs, and purified FKBP23 protein was shown to have Ca2+-binding ability. This is the first report of a Ca2+-binding FKBP. FKBP23 is a glycoprotein retained in the endoplasmic reticulum by its carboxyl-terminal tetrapeptide His-Asp-Glu-Leu, as demonstrated by immunostaining, retention, and deglycosylation assays. FKBP23 mRNA is expressed most strongly in heart, lung, and testis, beginning at day 8.5 of embryonic development. The FKBP23 gene was mapped to mouse chromosome 2.