We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions, the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the mechanisms and the control of DNA replication in eukaryotes.