Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-α is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-α levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-α peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-α in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-α peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-α-like immunoreactivity to be present in HeLa cells which released salusin-α-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-α. These results present the first evidence that salusin-α is biosynthesized and released from human-derived cells.