We have directly tested the hypothesis that elevated levels of dUTP cause the formation of small DNA fragments at the replication fork of Escherichia coli. Addition of increasing levels of dUTP to lysates on cellophane discs results in an increasing number of strand scissions in the newly replicated DNA. Lysates of strains defective in dUTPase produce many more scissions at the same level of dUTP. The size distribution of Okazaki pieces obtained in vivo can be reconstituted in vitro on cellophane discs if appropriate levels of dUTP are present. Although uracil excision leads to the apparent production of Okazaki pieces from both daughter strands, DNA synthesis is actually asymmetric under these conditions. De novo chain initiation events occur on only one strand. It is suggested that asymmetry of synthesis in vivo may be masked by uracil excision and other postreplication processing mechanisms.