Antibodies against pokeweed agglutinin binding proteins isolated from F9 embryonal carcinoma cells were used to screen a lambda gt11 expression library constructed from the cells. A cDNA clone thus obtained encoded a novel calcium binding protein of 140 kDa (CBP-140). Antibodies raised against the CBP-140 fusion protein stained a 140 kDa band in extracts not only from F9 cells but also from various mouse organs. A calcium blot experiment using CBP-140 fusion protein verified the calcium binding property of the protein. In the partial amino acid sequence so far clarified (652 amino acid residues) we could not detect EF-hand, but could detect contiguous acidic amino acids, which may serve as a calcium-binding site. CBP-140 showed homology with 70-kDa heat shock protein, though it was not induced by heat shock treatment. Localization of CBP-140 in endoplasmic reticulum was shown by indirect immunofluorescence staining and also by subcellular fractionation. Amino acid sequence of CBP-140 contains a carboxyl-terminal Asn-Asp-Glu-Leu (NDEL) sequence, which resembles Lys-Asp-Glu-Leu (KDEL) sequence, a signal to retain the resident proteins in endoplasmic reticulum; NDEL sequence may indeed play a similar role.