The cell nucleus is highly organized into distinct domains that spatially separate physiological processes. One of these domains, the Sp100-promyelocytic leukemia protein nuclear body (NB), is implicated in pathological processes, such as cancer and viral infection, yet its functions remain poorly understood. We show here that Sp100 interacts physically and functionally with ETS-1 and that NB morphology is affected by ETS-1. ETS-1 is a member of the ets family of transcription factors, which are key mediators of physiological and pathological processes. We have found that Sp100 interacts with two regions of ETS-1 (domains A+B and D+E+F). ETS-1 alters NBs while remaining localized throughout the nucleus, apparently by recruitment of the core component Sp100 away from the NBs. Sp100 strongly increases ETS-1 activation of natural and ets-focused promoters, through a mechanism involving the activation (C) domain of ETS-1 in addition to the interaction domains. Sp100 acts as a novel coactivator that potentiates the activator function of ETS-1. Our results provide an important new connection between nuclear structures and an important regulator of gene expression.