We previously proposed that a local duplication, not the loss of the subsequently amplified marker from its original site, might be the first step in gene amplification in human cells. It is important t o investigate this issue in naturally occurring amplification and when copy numbers are relatively low. We have examined the location of single-copy and amplified I I q I 3 sequences in cell lines from human breast cancers and squamous cell carcinomas using fluorescence in situ hybridization both with a probe specific for the I I q I 3 amplifying region and with a chromosome I I -specific library. We show that in most cell lines the 1 I q I 3 amplicons are physically linked t o chromosome I I or t o a chromosome derived from chromosome I I by various rearrangements near the I I q I 3 region. In none of the cell lines were interstitial deletions of I I q I 3 detected. These results indicate that I I q I 3 amplification in human tumor cells generally does not involve deletion as the initial step. One cell line with chromosomally located amplified I I q I 3 sequences contained double minutes that harbored the MYC gene but no I I q I 3 sequences. This suggests that the genetic outcome and the mechanism of gene amplification are probably dependent on specific DNA sequences rather than on the origin of the cells. Genes Cbrom Cancer 7: 74-84 (1993). 0 1993 Wiley-Liss, Inc.