Cells in which Alternative lengthening of telomeres (ALT) is activated have been reported to contain a novel PML nuclear body (PML-NB) known as ALT-associated PML body (APB). These large, donut-shaped nuclear structures are unique to telomerase-negative cells and contain, among others, PML, telomeric DNA, the telomere binding proteins TRF1 and TRF2, MRE11 and NBS1. APBs can be correlated with ALT in that they appear when the telomere length pattern becomes characteristic of ALT positive cells and are not found in telomerase positive cell lines. The exact role of these bodies in telomere maintenance and/or protection is as yet unclear as there have been few studies carried out to determine the functional involvement of the specific components. Our study aims at the identification of the essential components of this structure and, ultimately, at studying the effect of APB alterations on telomere length and genomic stability. We began by determining the percentage of APB-positive cells within our chosen cell lines, and found that there were a higher proportion of APB positive cells than reported in the literature. In order to determine the role of PML in formation of APB, we employed RNA interference oligonucleotides directed against nuclear PML. Interestingly, decreased speckled distribution of MRE11 and TRF1 was observed in PML-depleted cells. In contrast, TRF2 was not affected, and its down-regulation has no clear effect on MRE11 or TRF1 localization. TRF2 silencing results in telomere erosion, thus suggesting that in ALT cells TRF2 regulates telomere length. In addition, TRF2 depletion causes inhibition of colony formation, increased cell size, p21 upregulation and induction of genomic instability. Although in ALT cells active ATM is found at APBs, it is only following TRF2 silencing that the ATM target p53 becomes phosphorylated and activated. This study sheds new light on the role of the APB components PML and TRF2 in ALT cells.