Acylation-stimulating protein (ASP or C3adesArg) is a potent lipogenic factor in human and murine adipocytes and fibroblasts. The arginated form of ASP, i.e. complement C3a (C3a), stimulates immunological responses in human granulocytes, mast cells, guinea pig platelets and guinea pig macrophages; however, ASP is inactive in stimulating these responses. Thus both ASP and C3a are bioactive across species but are not functionally interchangeable. Tertiary structure of both proteins by X-ray crystallography and NMR spectroscopy predicts a tightly linked core region consisting of three α-helices linked via three disulphide bonds, with one of the α-helices extending out from the core and terminating in a flexible conformationally irregular carboxy-tail region. The present studies were undertaken in order to define the functionally active domains of ASP, distinctive from those of C3a, using chemical modifications, enzymic cleavage and synthetic peptide fragments. The results indicate that: (i) the N-terminal region (< 10 amino acids) plays little role in ASP receptor binding and triacylglycerol synthesis stimulation; (ii) the native C-terminal region had no activity, but modifications which increased hydrophobicity increased receptor binding, and led to some activation of triacylglycerol synthesis stimulation; (iii) an intact disulphide-linked core region is essential for triacylglycerol synthesis stimulation activity but not for receptor interaction. Finally, basic charges in the carboxy region (His) are essential for ASP triacylglycerol synthesis stimulation but not for receptor binding, whereas both functions are eliminated by the modification of Lys in the disulphide-linked core region. The present results suggest that there are two functional domains in AS, one that is responsible for the initial binding to the cell surface receptor, and a second domain that activates and increases triacylglycerol synthesis stimulation. This contrasts markedly with the structure-function studies of C3a where both binding competency and function were dependent on the C-terminal Arg. Thus ASP demonstrates distinct bioactivity.