Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.