The induction of ISG15 by interferon (IFN)-alpha/beta and subsequent protein ISGylation has been demonstrated in several cell types. However, regulation of free ISG15 levels and ISGylation by other IFNs and its implications in some carcinomas have not yet been completely evaluated. Here, we demonstrated that free ISG15 and ISGylation levels are enhanced by IFN-gamma treatment in the estrogen receptor-alpha-positive and -negative breast cancer cells, MCF-7 and MDA-MB-231, respectively. Specifically, IFN-gamma increases free ISG15 levels in the cytoplasm and ISGylation in the nucleus and cytoplasm, but in a manner distinct between MCF-7 and MDA-MB-231 cells. Therefore, free ISG15 and ISGylation may play central roles in mammary tumors by differentially modulating certain tumorigenic characteristics of estrogen receptor alpha-positive and -negative breast cancer cells. (C) 2018 Elsevier Inc. All rights reserved.