Horse heart cytochrome c was treated with methylsulfonylethyloxycarbonyl succinimide (Msc-ONSu) to give fully N(epsilon)-protected cytochrome c. Treatment of this derivative with a hard base for 15 sec regenerated the native tetrahectapeptide chain. CNBr degradation of the protected compound produced three fragments bearing only protective Msc functions on epsilon-amino groups. The fragment comprising the sequence 81-104 was isolated from the mixture and acylated with N-hydroxysuccinimidyl-t-butyloxycarbonyl-L-methioninate. The resulting pentacosapeptide derivative was partially deprotected by treatment with acid and condensed in good yield (65%) with fully synthetic N(alpha66), N(epsilon72,73,79)- tetra-Msc-cytochrome-c-(66-79)-tetradecapeptide azide. This pathway is preferred because the pentadecapeptide azide derivative 66-80 acylated the N(epsilon)-protected tetracosapeptide sequence 81-104 in an unpredictable manner. Subsequent treatment of the product with a base produced unprotected semisynthetic cytochrome-c-(66-104)-nonatriacontapeptide, which is known to undergo acylation by unprotected [Hse(65)]cytochrome-c-(1-65)-pentahexacontapeptide lactone. The high specificity of this condensation is ascribed to "conformation direction." Semisynthetic [Hse(65)]cytochrome c thus prepared reacts like native cytochrome c with a succinate cytochrome c reductase preparation and with cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). This semisynthetic strategy may provide a rapid route for the production of cytochrome c analogs modified in the highly conservative sequence 66-80.