Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR). Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.