Abstract: Endogenous N-sulfated glycosaminoglycans (GAGs) comigrating with standard heparin and sensitive to nitrous acid treatment were isolated from plasma of healthy donors. The amount of these compounds was 7-10 microg/ml, and activated partial thromboplastin time, anti-Xa and anti-IIa activities were similar to those of standard heparin of high molecular mass. Analysis with gradient PAGE of the putative endogenous heparin showed a mean molecular mass of 12 kD. These N-sulfated GAGs could be isolated only after removal of binding peptides that impaired purification by ion-exchange chromatography. We used SDS-PAGE as a tool to separate peptides from endogenous GAGs. N-sulfated GAGs exited the gel before peptides when the electrophoresis was overrun. Endogenous GAGs could be recovered by ion-exchange chromatography of the SDS-PAGE buffer, 'free' from associating peptides. These results strongly support the hypothesis that endogenous heparin is associated in vitro with a variety of proteins and that this association could be responsible for modification of both heparin and protein activities.