Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.