An important consideration in the construction of active and stable circularly permuted proteins is the connective sequence that links the native N- and C-termini. For this reason, various lengths of polyglycine linkers (two, three, four, five and six glycines) were employed to connect the original N- and C-termini of a circularly permuted construct of Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus was Met16. Examination of the circular dichroism (CD) spectra, gel-filtration chromatography elution profiles, urea-induced unfolding properties and enzyme kinetics revealed that, among the linkers tested, a linker length of five glycines was the most favorable. The Vmax of the circularly permuted variant with a five glycine linker (cpM16G5) was about 20% that of wild-type DHFR, although far UV CD spectra, gel filtration elution time, conformational stability and Km for the substrate dihydrofolate and Kd for the coenzyme NADPH were comparable in the two proteins. Another circularly permuted DHFR with a five glycine linker in which a new N-terminus was created at Leu24 (cpL24G5) was also constructed and assayed. The Vmax of cpL24G5 was almost the same as the wild-type, presumably due to the optimization of the glycine linker. The improved activity of the Leu24 permutant is probably due to the disruption of a catalytically important structure, the M20 loop, in the Met16 permutant.