The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-beta, ori-beta', and ori-gamma). We previously showed that deletion of the most active site or region (ori-beta) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-beta', both ori-beta and ori-beta', an 11-kb region just downstream from the DHFR gene, or the central approximately 40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur.