Herpesvirus saimiri-specific proteins from the nuclear fractions of productively infected owl monkey kidney cells were dissociated from virus and host DNA by treatment with 2 M-NaCl or separation on Urografin density gradients. Empty virus capsids remained intact and could be separated from major non-structural proteins (110K, 51K and 48K) and from a subset of structural proteins (130K, 29K and 12K), either by Urografin gradient sedimentation or differential centrifugation. The DNA in such soluble extracts of nuclear proteins was efficiently removed by spermine precipitation, together with the host cell histones and large fractions of the 130K and 12K structural proteins. Proteins in the spermine-soluble fraction were analysed by affinity chromatography on columns of single-stranded calf thymus DNA coupled to cellulose. Two major structural proteins (130K and 12K), whose synthesis was sensitive to phosphonoacetic acid (PAA), and one minor PAA-resistant structural protein (29K) bound to DNA-cellulose. The major PAA-resistant 110K non-structural protein and the PAA-resistant non-structural 51K and 48K phosphoproteins were efficiently released into the spermine-soluble fraction and also bound to DNA-cellulose as did the 76K protein and minor species of 42K, 39K, 34K, 25K and 21K. Virus-specific proteins were eluted from such columns by buffers containing 0.4 M-NaCl or by heparin in low-salt buffers. Polypeptides from virus particles, infected cell extracts, or samples of eluates from DNA-cellulose chromatography, were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose filters and probed for their ability to bind labelled polynucleotides. The non-structural 51K phosphoprotein, the 12K and 29K structural proteins and a 100K virion polypeptide all bound labelled DNA. However, the binding activities of the 130K protein from virions or purified by affinity chromatography and of the 110K polypeptide could not be demonstrated reproducibly after transfer from SDS gels to nitrocellulose. Comparisons of the present results on the properties of the herpesvirus saimiri-specified DNA-binding proteins with published accounts of the DNA-binding proteins of other herpesviruses, suggest some striking similarities with the DNA-binding proteins of the Epstein-Barr virus.