Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway. We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases. In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of nonsense transcripts. To distinguish stabilized nonsense transcripts from background transcripts upregulated by drug treatment, drug-induced expression changes are measured in control and disease cell lines with complementary DNA (cDNA) microarrays. Transcripts are ranked by a nonsense enrichment index (NEI), which relates expression changes for a given transcript in NMD-inhibited control and patient cell lines. The most promising candidates can be selected using information such as map location or biological function; however, an important advantage of the GINI strategy is that a priori information is not essential for disease gene identification. GINI was tested on colon cancer and Sandhoff disease cell lines, which contained previously characterized nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively. A list of genes was produced in which the MLH1 and HEXB genes were among the top 1% of candidates, thus validating the strategy.