Alternative oxidase (AOX) has been found in a large number of filamentous fungi and yeasts with the notable exceptions of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In virtually all of these fungi, AOX is induced by stresses on the cell that compromise the efficiency of the standard mitochondrial electron transport chain. As AOX is encoded in the nucleus and the signals that induce its expression originate in mitochondria, induction of the enzyme provides a classic example of retrograde regulation where signals from mitochondria influence the expression of nuclear genes. We have previously isolated mutants in Neurospora crassa that are incapable of inducing AOX. The genes affected in two of these mutants, aod-2 and aod-5, encode zinc cluster transcription factors that act to control expression of the AOX by binding to an alternative oxidase induction motif (AIM) found in the promoter of the AOX structural gene. We have now used pull-down assays and size-exclusion chromatography to demonstrate that the AOD2 and AOD5 proteins physically interact in vitro. In addition, we have shown that a homolog of the RTG2 protein, which is part of a classic retrograde signaling pathway in S. cerevisiae, is not required for AOX regulation in N. crassa.