Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na+-Ca2+ exchange activities with a goal to identify a potent blocker. The cyclic hexapeptides, having the different amino acid sequence, contain two arginines (to retain a positive charge), two phenylalanines (to control hydrophobicity), and two cysteines (to form an intramolecular S-S bond). The effect of cyclic hexapeptides were tested on Na+-Ca2+ exchange and its partial reaction, the Ca2+-Ca2+ exchange, by measuring the 45Ca fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven cyclic hexapeptides inhibit Na+-Ca2+ exchange with a different potency (IC50 = 2-300 μM). Phe-Arg-Cys-Arg-Cys- Phe-CONH2 (FRCRCFa) inhibits the Na+(i)-dependent 45Ca uptake (Na+- Ca2+ exchange) and Ca2+(i)-dependent 45Ca uptake (Ca2+-Ca2+ exchange) in the isolated cardiac sarcolemma vesicles with IC50 = 10 ± 2 μM and IC50 = 7 ± 3 μM, respectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a 'slow' binding (a maximal inhibitory effect is already observed after t = 1 s of mixing). The inside positive potential, generated by Na+(o)-dependent Ca2+ efflux, was monitored by Oxanol-V (A635-A612) or Merocyanine-540 (A570-A500). In both assay systems, FRCRCFa inhibits the Na+-Ca2+ exchange with IC50 = 2-3 μM, while a complete inhibition occurs at 20 μM FRCRCFa. The forward (Na+(i)-dependent Ca2+ influx) and reverse (Na+(o)-dependent Ca2+ efflux) modes of Na+-Ca2+ exchange, monitored by Arsenazo III (A600- A785), are also inhibited by FRCRCFa. The L-Arg4 → D-Arg4 substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na+](i) (160 mM) or [Ca2+](i) (250 μM) and varying 45Ca(o) (2- 200 μM), FRCRCFa decreases V(max) without altering the K(m). Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca2+ either for Na+-Ca2+ or Ca2+-Ca2+ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding.