The pyrimidine nucleobase analogue6H, 8H-3,4-dihydropyrimido[4,5-c]-[1,2]oxazin-7-one (P) is a mimic both of cytosine and thymine, since it can form stable hydrogen-bonded base-pairs with either guanine or adenine. To investigate the geometric properties of pairing with guanine in a DNA double helix, the structure of d(CGCGPG) 2 has been determined by single crystal X-ray analysis. The oligonucleotide crystallised as a left-handedZ-DNA duplex in the orthorhombic space groupP2 1 2 1 2 1 with cell dimensionsa= 18.23 Å,b= 30.63 Å,c= 43.78 Å. Refinement using NUCLSQ with 51 water molecules included in the final model converged atR= 0.179 (R w = 0.159) for 2798 reflections (F2σ(F)) in the range 8 Å to 1.7 Å. Remarkably, the two P.G pairs in the hexamer duplex are different: Watson-Crick and wobble types separately illustrate both cytosine-like and thymine-like behaviour. The result suggests that mutagenesis experiments involving P and other analogues which display pronounced base-pairing ambivalence can be used to examine the structural basis of substrate discrimination by polymerases that is essential to accurate genetic replication.