Short DNA chains, isolated from in vitro pulse-labeled replicating polyoma DNA, exhibit some degree of self-complementarity (28% resistance to S1 nuclease after self-annealing to plateau levels). This level of self-annealing is not increased if short DNA chains present as free single-stranded DNA after extraction are included in the hybridization, excluding a selective loss of chains from one side of the growing fork and supporting a semi-discontinuous mode of chain growth. This mode also applies to restricted synthesis conditions under which a relative excess of short chains is made, since no increase in the self-annealing of such short chains is observed. The self-annealing that can be measured is higher for the faster sedimenting portion (46%) of the short DNA chains than for the slower sedimenting portion (18.5%), indicating that it is most likely due to contaminating continuously growing strands from the other side of the fork. High self-annealing values (up to 60%) are obtained if virus stocks generating defective DNA are used for infection. Restriction endonuclease (Hpall) characterization of such DNA shows evidence for the presence of multiple origins of replication. One of several possible mechanisms is discussed by which replicating defective DNA can generate self-complementary short chains despite a semi-discontinuous mode of replication.