The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.