Acylation Stimulating Protein (ASP) is a human plasma protein that stimulates both triacylglycerol synthesis and glucose transport. ASP is identical to C3adesArg and is generated by the interaction of factor B, complement C3 and adipsin. We have demonstrated that mature fat cells express messages for factors B, complement C3 and adipsin; that human pre-adipocytes, when cultured under differentiating conditions to produce adipocytes, generate ASP in the culture medium; and that human adipocytes also become more responsive to ASP as they differentiate. The aim of this study, therefore, was to examine the temporal production of ASP during adipocyte differentiation in relation to other adipose specific factors involved in lipogenesis. The results demonstrate that (i) there was little ASP production by differentiating adipocytes over the first 7 days, with a marked increase in ASP thereafter (up to sixfold); (ii) this increase was paralleled by large increases in the message level of factor B and complement C3 and moderate increases in adipsin message; (iii) increases in lipoprotein lipase (LPL) message and glycerol-3-phosphate dehydrogenase (GPDH) activity (both key enzymes for substrate supply for triacylglycerol synthesis) occurred earlier than the increase in ASP; and (iv) in spite of the increase in LPL and GPDH, triacylglycerol synthetic capacity only markedly increases following the increase in ASP production in adipocytes. Although the present study cannot be interpreted as showing causality with respect to triacylglycerol synthesis, it does point to an important role for ASP in human adipose tissue physiology.