Background. Transplantation tolerance is induced by perioperative administration of host class I major histocompatibility complex proteins bearing donor-type amino acid (a.a.) epitopes substituted far native residues. Herein we demonstrate that two cryptic tolerogenic a.a. epitopes are localized in the alpha(1)-helical region of the rat RT1.A(u) class I major histocompatibility complex alloantigen. Methods. Three allochimeric proteins were produced by superimposing the nucleotides encoding donor-type RT1.A(u) a.a. onto the host RT1.A(a) backbone using the polymerase chain reaction-based method of gene splicing with overlap extension. We substituted nucleotide sequences encoding all nine (Arg(62), Glu(63), Gln(65), Gly(66), Gly(69), His(70), Val(73), Asn(74), and Asn(77); alpha(1h) (u)62-77-RT1.A(a)), the first four (Arg(62), Glu(63), Gln(65), and Gly(69); alpha(1h) (u)62-69-RT1.A(a)), or the last four (His(70), Val(73), Asn(74), and Asn(77); alpha(1h) (u)70-77-RT1.A(a)) alpha(1)-helical RT1.A(u) polymorphic a.a. in RT1.A(a) cDNA. A baculovirus/Spodoptera frugiperda (Sf9) expression system was harnessed for production of the proteins. Results. Untreated ACI (RT1(a)) rats reject Wistar-Furth (WF; RT1(u)) heart allografts at a mean survival time of 8.9 +/- 1.0 days. A single portal vein injection of 10 mu g of alpha(1h) (u)62-77-RT1.A(a) protein had no effect on the survival of WF heart allografts (10.5 +/- 0.6 days) in ACI hosts. Interestingly, portal vein administration of 10 mu g of alpha(1h) (u)70-77-RT1.A(a) induced transplantation tolerance toward WF grafts in four of six untreated ACI recipients (>100 days; P<0.01). In contrast, 10 mu g of alpha(1h) (u)62-69-RT1.A(a) only prolonged the survival of WF heart allografts in ACI hosts (14.0 +/- 0.8 days; P<0.01). However, when combined with a 7-day course of cyclosporine (4.0 mg/kg; oral gavage), alpha(1h) (u)62-69-RT1.A(a) induced tolerance toward WF allografts in five of seven ACI recipients (>170 days; P<0.01). Long-term survival of WF grafts was not achieved when a 7-day course of cyclosporine was administered alone (14.3 +/- 3.0 days) or with 10 mu g of alpha(1h) (u)62-77-RT1.A(a) (14.6 +/- 0.6 days; NS). Conclusions. These findings suggest that the use of allochimeric proteins may provide a novel approach to the induction of tolerance.