Serial undiluted passage of polyoma virus derived from transfection of mouse fibroblasts with well defined wild-type genomes results in the appearance of a very heterogeneous population of defective virus particles. Many of these variants show duplications which not only contain the cis-acting region minimally required for efficient replication, but also other regions. A detailed analysis of the duplication patterns appearing in high multiplicity infections is presented. We performed heteroduplex analyses of duplicated fragments using mung bean nuclease and demonstrated that the pattern of duplication junctions is conserved qualitatively and quantitatively. However, the distribution of fragment sizes varied in a number of independently derived virus stocks. Amplification of viral nucleotide sequences is an early event in virus replication, occurring at least as early as 3 days post-transfection. The pattern of duplication did not change significantly in successive early passages at high multiplicity. Although duplication was accompanied by deletion of various parts of the viral genome, a sequence bordering the duplications at the late side of the origin of replication was retained as a single copy in all defective viruses. The relevance of these findings to the mechanism that creates the duplications and the biological activity of defective virus is discussed.