The 24-kDa band formed when sera of humans infected with human T cell lymphotropic virus type I (HTLV-1) were reacted with HTLV-I lysates in conventional Western blot (WB) assays was found to be composed of two immunologically unrelated proteins of 24- and 23-kDa. p24, but not p23, carries epitopes shared by the major core proteins of the other known transactivating C-type retroviruses. p23 is unrelated immunologically to the env and tax HTLV-I products but partly cross-reacts with HTLV-I p19. All HTLV-I and simian T cell leukemia virus type I sera tested reacted with p23. Reactivity with p23 was seen with some HTLV-I sera that did not react or reacted weakly with HTLV-I p24. No reactivity with p23 was seen among the 51 human HTLV-II sera tested nor among a large panel of control sera. Because of its type-specificity and strong immunogenicity, p23 provides a reliable serologic marker for the diagnosis of HTLV-I infection and for distinguishing between HTLV-I and -II.