In the yeast S. cerevisiae, ARS (autonomously replicating sequence) elements located in the intergenic spacers of the rRNA gene locus are infrequently activated as origins of replication. We analyzed the rARS activation with a combination of neutral/neutral (N/N) two-dimensional (2D) gel electrophoresis and either the intercalating drug psoralen, which in vivo specifically marks the transcribing gene copies, or the selective accessibility of restriction sites in transcriptionally active genes. We found that initiation of replication starts at those rARSs placed immediately downstream of transcribing rRNA genes. This correlation between transcription and replication is consistent with the presence of nucleosome-free enhancers at each transcriptionally active gene copy and suggests that the transcription factor Abf1p is involved in replication initiation at the ARS in the rDNA gene locus.