Background: Human mitochondrial complex I is composed of 44 subunits with an elaborate assembly mechanism. Results: TALENs were used to knock out complex I subunit NDUFA9 in HEK293T cells, revealing a novel membrane arm subcomplex. Conclusion: NDUFA9 is critical for stabilizing the junction between different arms of complex I. Significance: TALEN-mediated knock-out represents an accessible approach to address protein function in human cells. Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture ofNDUFA9cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9contained a complex I subcomplex consisting ofmembrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.