Initiation of DNA replication in metazoan, in general, occurs within specific initiation zones. The metazoan rRNA genes (rDNAs) are arranged in tandem repeated units and replication of the rDNA initiates within the intergenic spacer (IGS) in adult tissues and late stage embryos. However, in embryos undergoing rapid cell division and in extracts from these embryos, rDNA replication has been reported to initiate at multiple, random sites throughout the rDNA. We performed time course analyses of initiation of Xenopus laevis rDNA replication in Xenopus egg extracts by two-dimensional (2D) agarose gel electrophoresis. We found that the first initiation events in rDNA occur preferentially in the intergenic spacer and that, later on, initiation events can occur at multiple sites throughout the rDNA.