We isolated a cDNA encoding the rat membrane-associated 65/67-kDa calcium-binding protein, CBP 65/67, from a lambda ZAP II cDNA-expression library of rat liver by immunoscreening using monospecific polyclonal anti-(CBP 65/67) antibodies and monoclonal anti-(CBP 65/67) IgG. The product of this cDNA expressed in Escherichia coli was confirmed as CBP 65/67 both by immunostaining and by comparison of the molecular mass with the CBP 65/67 isolated from rat liver by SDS/PAGE. The cDNA sequence and the deduced amino acid sequence of CBP 65/67 both show a high degree of identity to human p68 and human calelectrin, which belong to a family of calcium-dependent, membrane-associated, phospholipid-binding proteins, called annexins. This means that CBP 65/67 is a homolog of the two human proteins just mentioned above. We are not aware that a rat annexin VI has previously been isolated and sequenced. The mRNA expression of CBP 65/67 in different rat organs during development was investigated by Northern blot analysis. In adult tissues, high mRNA levels of CBP 65/67 were found in lung, heart, muscle, spleen and especially in thymus and pancreas, whereas in liver, kidney, intestine, stomach and brain only low levels of CBP 65/67 mRNA could be detected. The amount of mRNA during tissue development in kidney, stomach and muscle showed only slight changes. In contrast, a significant increase of CBP 65/67 expression was observed in liver, lung, heart and brain. In most of the organs investigated, the level of mRNA correlated closely with the level of protein expression, indicating that the expression of CBP 65/67 in most organs is controlled primarily at the transcriptional level.