Cyclic phosphodiesterase and 2'-5' RNA ligase are members of a superfamily of proteins which share structural similarities even though their homology may be very low. A putative 2'-5' RNA ligase from Pyrococcus horikoshii has been crystallized and its X-ray crystallographic structure determined to 2.4 A. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.07, b = 45.47, c = 93.17 A and one protein monomer in the asymmetric unit. The molecular-replacement probe was a 2'-5' RNA ligase from Thermus thermophilus which shares 30% sequence identity. The P. horikoshii RNA ligase has some structural features that have more in common with a cyclic phosphodiesterase from Arabidopsis thaliana with which it has no significant homology, yet an examination of the electrostatic surface potential clearly defines its relationship to the T. thermophilus RNA ligase. However, the size of the active-site cleft is smaller and less positively charged than that of the T. thermophilus homologue, suggesting that the actual substrate may be smaller than that previously postulated for the latter.