Abstract A high-density lipoprotein (HDL) fraction was isolated from the hemolymphatic plasma of the spider <i>Polybetes pythagoricus</i> by density-gradient ultracentrifugation. Hydrated density (1.13 g/ml), electrophoretic mobility (SDS—PAGE) of apoproteins and lipid classes composition were determined. Lipids were identified by HP-TLC and auxiliary techniques; they were quantified by TLC-FID. The protein moiety is composed of two main apoproteins (250 and 76 kDa, respectively) and several polypeptides of low molecular weight. It resembles the apoliphorins of insects and some other arachnids. The lipid composition differs from most lipophorins. Phospholipids amount to more than 60% of total lipids, while diacylglycerols (2.4%) are supplanted by triacylglycerols (16.5%) as the main circulating energetic lipids.Facultad de Ciencias Naturales y MuseoInstituto de Investigaciones Bioquímicas de La Plata