The ssh10b and ssh10b2 genes, a pair of distantly related paralogues in Sulfolobus shibatae, encode members of the Sac10b DNA binding protein family in thermophilic archaea. It has been shown previously that Ssh10b exists in abundance in S. shibatae and is capable of constraining negative DNA supercoils, properties that are consistent with a speculated architectural role for the protein in chromosomal organization. In this study, the ssh10b2 gene was cloned and expressed in Escherichia coli, and the recombinant Ssh10b2 protein was purified to apparent homogeneity. Immunoblotting analysis using a specific anti - Ssh10b2 antibody showed that ssh10b2 was expressed in S. shibatae, but the cellular level of Ssh10b2 was only - 10% of that of Ssh10b. Recombinant Ssh10b2 was capable of interacting with both double-stranded and single-stranded DNA. The affinity of the protein for double-stranded DNA was higher than that reported for Ssh10b. The Ssh10b2 and Ssh10b proteins appeared to generate similar gel shift patterns on duplex DNA fragments. However, unlike Ssh10b, Ssh10b2 was unable to constrain DNA supercoils. These data suggest that Ssh10b2 does not serve as a general architectural factor in DNA compaction and organization in S. shibatae.