We describe here the use of chromatin as a target for retroviral integration in vitro. Extracts of cells newly infected with murine leukemia virus (MLV) provided the source of integration activity, and yeast TRP1ARS1 and SV40 minichromosomes served as simple models for chromatin. Both minichromosomes were used as targets for integration, with efficiencies comparable with that of naked DNA. In addition, under some reaction conditions the minichromosomes behaved as if they were used preferentially over naked DNAs in the same reaction. Mapping of integration sites by cloning and sequencing recombinants revealed that the integration machinery does not display a preference for nucleosome-free, nuclease-sensitive regions. The distributions of integration sites in TRP1ARS1 minichromosomes and a naked DNA counterpart were grossly similar, but in a detailed analysis the distribution in minichromosomes was found to be significantly more ordered: the sites displayed a periodic spacing of approximately 10 bp, many sites sustained multiple insertions and there was sequence bias at the target sites. These results are in accord with a model in which the integration machinery has preferential access to the exposed face of the nucleosomal DNA helix. The population of potential sites in chromatin therefore becomes more limited, in a manner dictated by the rotational orientation of the DNA sequence around the nucleosome core, and those sites are used more frequently than in naked DNA.