The cDNA of a Ca(2+)-binding protein regucalcin was cloned from a rat liver cDNA library which was constructed in lambda ZAPII by immunoscreening. Positive clones were obtained from which spanned the region of interest, and they gave a sequence of 1.7 kb by sequencing with the dideoxynucleotide method. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the regucalcin molecule. Regucalcin was composed of 299 amino acid residues and its molecular weight was estimated to be 33,388 Da. The hydropathy profile of regucalcin showed a highly hydrophilic character. The nucleotide and amino acid sequences of regucalcin did not have statistically significant homology, as compared with the registered sequences which are found in the EMBL and GenBank databases containing several other Ca(2+)-binding proteins (calmodulin, calbindin-D28k and S-100 beta). The regucalcin molecule did not contain the EF-hand motif as a Ca(2+)-binding domain. The present study demonstrates that regucalcin is a unique Ca(2+)-binding protein in the liver of rats.