Abstract Box-Asp-ϵAhx-Ser-ϵAhx-His-ϵAhx-OEt(Linear-6), cyclo(-Asp-ϵAhx-Ser-ϵAhx-His-ϵAhx-)(Cyclic-6) and poly(-Asp-ϵAhx-Ser-ϵAhx-His-ϵAhx-)(Poly-6) were synthesized, and their catalytic actions in the hydrolysis of PNPA were investigated. Linear-6 was prepared by fragment condensations of three peptides having the sequence -Asp(OBzl)-ϵAhx-, -Ser(Bzl)-ϵAhx- and -His-ϵAhx-, respectively, and subsequent debenzylation. Cyclic-6 and Poly-6 were obtained by cyclization and polymerization, respectively, of H-Asp(OBzl)-ϵAhx-Ser(Bzl)-ϵAhx-His-ϵAhx-OH with DPPA, and subsequent deprotections. The reaction velocities in the hydrolysis of PNPA were all proportional to [E] or [S], and all peptides gave the bell-shaped pH- κ cat profiles having optima around pH 8.2. The reaction velocity of Cyclic-6 was always larger than that of Linear-6 or Poly-6. The velocities of all reactions increased steadily with rise in temperature, and the Arrhenius' plots from T- k cat relations suggested that the activation energy for the reaction catalysed by Poly-6 is larger than that by Linear- or Cyclic-6. Brief results for the hydrolysis of other substrates, the solvent isotope effect, and the conformational study with c.d. measurements are also reported.