Nuclear respiratory factor (NRF)-1 appears to be important for the expression of several respiratory genes, but there is no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of doxycycline, UCP-1 mRNA and protein were undetectable. In the presence of doxycycline, UCP-1 was expressed and oxygen consumption doubled. This rise in oxygen consumption was associated with an increase in NRF-1 mRNA. It was also associated with an increase in NRF-1 protein binding activity as determined by electrophoretic mobility shift assay using a functional NRF-1 binding site from the δ-aminolevulinate (ALA) synthase promoter. Respiratory uncoupling also caused a time-dependent increase in protein levels of ALA synthase, an early marker for mitochondrial biogenesis. ALA synthase induction by respiratory uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the region of the NRF-1 initiation codon; a scrambled oligonucleotide with the same base composition had no effect. Respiratory uncoupling increases oxygen consumption and lowers energy reserves. In HeLa cells, uncoupling also increases ALA synthase, an enzyme critical for mitochondrial respiration, but only if translatable mRNA for NRF-1 is available. These data suggest that the transcription factor NRF-1 plays a key role in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy.