Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide, which causes widespread human and animal diseases. The need for new therapeutic agents along with the biology of these parasites has fueled a keen interest in the understanding of the nutrients acquisition by these parasites. Studies on the characterization of the T. gondii cyst wall as well as the contribution of the host cell to this formation have been little explored. The aim of this paper was to investigate the electric surface charge of the T. gondii tissue cysts by ultrastructural cytochemistry, through polycationic markers, employing ruthenium red (RR) and cationized ferritin (CF). Glycosaminoglycans revealed by RR were localized on the cyst wall as a homogeneous granular layer electrondense, all over its surface. The incubation of living tissue cysts with CF for 20 min at 4 degrees C followed by the increase of temperature to 37 degrees C indicated that T. gondii cyst wall is negatively charged and that occurs an incorporation of anionic sites by the cyst wall, through vesicles and tubules, and their posterior location in the cyst matrix. So, as to identify which group of molecules produces negative charge in the cyst wall, we used enzymes for cleavage on different types of molecules, demonstrating that the negative charge in the cyst wall is mainly produced by phospholipids. Our results, described in this work show, for the first time, the negativities of the cyst wall, the incorporation and the traffic of intracellular surface molecules by T. gondii cyst wall. Our model of study can give an important contribution to the knowledge of the biology and the processes involved in nutrients acquisition by bradyzoites living inside the cysts and, and also be applied as a target for the direct action of drugs against the cyst.