We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes. (41) showed that DNA replication initiates at multiple locations in a plasmid containing a 20-kb human chromosomal sequence in human cells. Existence of multiple origins of replication was also suggested for the amplified chorion genes in Drosophila cells by the 2D gel electrophoretic method (20,32).In contrast to these results, evidence for specific replication origins in higher eukaryotes has also been reported. The most striking contradiction is a discrepancy between the data on replication origins in the DHFR locus of CHO cells obtained by different experimental approaches. Although the 2D gel analysis of replication intermediates suggested the presence of multiple initiation sites distributed in a broad initiation zone as mentioned above, other workers reported that replication in this chromosomal region initiates at a specific site, as judged from nascent strand analyses (15,16,55