The broad-host-range plasmid pAM beta 1 from Gram-positive bacteria encodes a resolvase, designated Res beta, which shares homology with the proteins of the resolvase-invertase family. Here we report the purification and in vitro characterization of Res beta. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro. Res beta binds to two regions within its resolution site res. One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed.