We recently identified a region of preferential replication initiation, oriGNAI3, near the 3' end of the Chinese hamster GNAI3 gene. oriGNAI3 is co-amplified in mutants selected for AMPD2 amplification, a process generating chromosomal rearrangements. In this report we have taken advantage of cell lines with truncated and translocated amplified units to show that these rearrangements do not alter the function of ori GNAI3. These results indicate that replication initiation at this locus relies essentially on local features. Interestingly, the study of one line in which a rearrangement has disrupted the GNAI3 gene shows that ongoing transcription of this gene is not required for initiation at oriGNAI3. In order to obtain further insight into the sequences and/or chromatin structures required for oriGNAI3 function, we have analyzed the DNase I sensitivity and nucleotide sequence of the region. The features important for replication initiation appear to cluster in a 7-12 kb region which includes oriGNAI3.