We investigated the effects of [pGlu4,Cyt4]AVP(4–9) fragments and its analogues on cycloheximide CHX-induced learning impairment in rats using the step-through-type passive avoidance test in rats. CHX (2.8 mg/kg, s.c.) significantly shortened the step-through latency in the retention trial. pGlu–Asn–Cys(Cys)–Pro–Arg–Gly–NH2 ([pGlu4,Cyt6]AVP(4–9); 10 ng/kg, s.c.), a major metabolite of arginine vasopressin, improved the CHX-induced learning impairment. Asn–Cys–Pro–Arg–OH ([Cys6]AVP(5–8); 1 ng/kg) corrected avoidance learning in the CHX-treated group, whereas neither Cys(Cys)–Pro–Arg–OH nor pGlu–Asn–Cys(Cys)–Pro–OH had any effect (1, 10 and 100ng/kg, s.c.). pGlu–Asn–Ser–Pro–Arg–Gly–NH2 (No. 302), a newly synthesized [pGlu4,Cyt6]AVP(4–9) analogue, significantly prolonged the latency shortened by CHX at doses of 0.1, 1 and 10 ng/kg (s.c.). Asn–Ser–Pro–Arg–OH also improved the learning disruption induced by CHX, although the effective dose was 100 times higher than that of No. 302. The half-life of No. 302 in rat blood was about 5.5, 22 and 25 times longer than that of [pGlu4,Cyt6]AVP(4–9); ([Cys6]AVP(5–8); and Asn–Ser–Pro–Arg–OH, respectively. These results suggest that [Cyst6]AVP(5–8) is the minimal effective amino acid sequence in [pGlu4,Cyt6]AVP(4–9), and show that No. 302 is a potent, pharmacologically active peptide with high stability in the blood.