Resistance to lysis by human serum (HS) is an important parameter used to distinguish Trypanosoma brucei brucei from both Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Neither the exact nature of the trypanolytic factor (TLF) nor the mechanism of action by which HS lyses susceptible trypanosomes is well understood. This report tries to elucidate the role played by the variable surface glycoprotein (VSG) coat and trypanosome surface-related processes in the mechanism of HS lysis of HS-sensitive (HSS) and HS-resistant (HSR) trypanosomes. Procyclic forms of T. brucei gambiense transformed from either HSS or HSR bloodstream stages were found to be HSR. These procyclic forms were shown to have lost their VSG coat. However, the addition of excess soluble VSG from HSS trypanosomes did not block lysis of HSS trypanosomes. Human serum lysis was significantly inhibited if the trypanosomes were incubated with membrane stabilizers, i.e., including cytochalasins (B, D, and E specifically), zinc acetate, vinblastine, and benzyl alcohol, or with the lysosomotropic agents ammonium chloride and chloroquine. The inhibition exerted by these compounds was always reversible. The results in this report, taken together, strengthen the hypothesis that the lytic factor interacts with and moves along the trypanosome surface to be internalized eventually.