Nuclear respiratory factor-1 is a transcriptional activator that has been implicated in the nuclear control of respiratory chain expression. Yeast two-hybrid screens were performed to identify proteins that physically interact with nuclear respiratory factor-1. Saturation screening of both mouse embryo and mouse testis libraries yielded 14 independent clones, all of which represented two different isoforms of dynein light chain. In addition to using the two-hybrid method, the specificity of the nuclear respiratory factor-1/dynein light chain interaction was established by chemical crosslinking of the purified native proteins and by co-immunoprecipitation of nuclear respiratory factor-1 and dynein light chain from mammalian cells. Both two-hybrid and chemical crosslinking assays demonstrated that binding of dynein light chain required the first 26 amino acids of nuclear respiratory factor-1. Although dynein light chain is associated with dynein, a cytoplasmic motor molecule, immunolocalizations showed substantial nuclear staining using several different anti-dynein light chain antibodies. Moreover, fluorescence overlays of confocal images established that nuclear respiratory factor-1 and dynein light chain displayed a very similar nuclear staining pattern. The significance of the nuclear respiratory factor-1/dynein light chain interaction was investigated further by determining whether a similar interaction was conserved between dynein light chain and the erect wing gene product of Drosophila, a protein related to nuclear respiratory factor-1 through its DNA binding domain. Here, we establish that the erect wing gene product can bind and trans-activate transcription through authentic nuclear respiratory factor-1 binding sites. Moreover, the erect wing gene product, like nuclear respiratory factor-1, interacted specifically with dynein light chain both in vitro and in transfected cells. Thus, the interaction with dynein light chain is conserved between transcription factors that are structurally and functionally similar between humans and Drosophila.