Epigenetic pathways help control the expression of genes. In cancer and other diseases, aberrant silencing or overexpression of genes, such as those that control cell growth, can greatly contribute to pathogenesis. Access to these genes by the transcriptional machinery is largely mediated by chemical modifications of DNA or histones, which are controlled by epigenetic enzymes, making these enzymes attractive targets for drug discovery. Here we describe the characterization of a locus derepression assay, a fluorescence-based mammalian cellular system which was used to screen the NCI structural diversity library for novel epigenetic modulators using an automated imaging platform. Four structurally unique compounds were uncovered that, when further investigated, showed distinct activities. These compounds block the viability of lung cancer and melanoma cells, prevent cell cycle progression, and/or inhibit histone deacetylase activity, altering levels of cellular histone acetylation.