Micrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-I) DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32P- or 3H-thymidine-labelled HSV-I. Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-I-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-I DNA as well as to 32P-labelled Vero cell DNA.. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with PH 9.3 buffer, pyridine, Sarkosyl or NcCl/urea. Only fragments of heterogeneous size were produced. Thus, HSV-I DNA is found predominantly in structures other than nucleosomes during virus replication.