Since de-novo synthesis of pyrimidine nucleotides is coupled to the mitochondrial respiratory chain (RC) via dehydroorotic acid dehydrogenase (DHODH), respiratory chain dysfunction should impair pyrimidine synthesis. To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human keratinocytes and 143B cells, a human osteosarcoma cell line, in culture. This resulted in severe impairment of de novo pyrimidine nucleotide synthesis. The effects of RC inhibition were not restricted to pyrimidine synthesis, but concerned purine nucleotides, too. While the total amount of purine nucleotides was not diminished, they were significantly broken down from triphosphates to monophosphates, reflecting impaired mitochondrial ATP regeneration. The effect of Rotenone was similar to that of Antimycin A. This was surprising since Rotenone inhibits complex I of the respiratory chain, which is upstream of ubiquinone where DHODH interacts with the RC. In order to avoid unspecific effects of Rotenone, we examined the consequences of a mitochondrial DNA mutation that causes a specific complex I defect. The effect was much less pronounced than with Rotenone, suggesting that complex I inhibiton cannot fully explain the marked effect of Rotenone on pyrimidine nucleotide synthesis.