The effect of allogeneic presensitization on Hya isograft survival was reinvestigated in C57BL, C3H and (C57BL x C3H)F1 females. Three weeks after transplantation of H-2 or non-H-2 differing female or male skin grafts, a syngeneic male skin graft (Hya isograft) was transplanted onto female recipients. C57BL and (C57BL x C3H)F1 females pretreated with a female skin graft of any origin rejected the C57BL male isograft as a first-set, while females pretreated with a male skin graft rejected the C57BL male isograft in a second-set manner. The same happened with the C3H male skin graft transplanted onto previously challenged (C57BL x C3H)F1 females. C3H females pretreated with a C3H or CBA female graft rejected the C3H male isograft as a first-set, whereas those pretreated with a C3H or CBA male graft rejected the male C3H isograft in a second-set manner. However, after pretreatment with a BALB/c male skin graft, C3H females were not sensitized to Hya. They rejected the C3H male isograft slightly faster than was expected for the first graft but this accelerated rejection was not specific, since it occurred as well in C3H females pretreated with grafts of BALB/c females. The best explanation for this unsuccessful immunization of C3H females is that an inferior, non-H-2-dependent Hya recognition system is involved in male isograft rejection.