For investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron-transport chain, ethoxy-substituted ubiquinone derivatives, 2-ethoxy-3-methoxy- or 3-ethoxy-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone (EtOQ0C10) and 2,3-diethoxy-5-methyl-6-decyl-1,4-benzoquinone [(EtO)2Q0C10], were synthesized and characterized. These compounds were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q0C10) by reaction with sodium ethoxide/ethanol in hexane under anaerobic conditions. The products, EtOQ0C10 and (ETO)2Q0C10, were separated by thin-layer chromatography using hexane/ether (3.5:1) as the developing solvent. The Rf values for diethoxy and monoethoxy derivatives are 0.7 and 0.6, respectively. The spectral and redox properties of EtOQ0C10 and (ETO)2Q0C10 are very similar to those of Q0C10. The reducibility of these derivatives by succinate was measured with succinate-Q reductase (SQR), and their oxidizability was measured by ubiquinol-cytochrome c reductase (QCR). Ethoxy ubiquinone derivatives exhibit concentration-dependent inhibition of SQR activity, with (ETO)2Q0C10 being the more potent inhibitor. These derivatives do not inhibit QCR and are reduced by succinate-cytochrome c reductase in an antimycin-insensitive manner. When used as substrate for QCR, EtOQ0C10H2 has about 55%, and (ETO)2Q0C10H2 about 15%, of the activity of Q0C10H2, but with lower apparent Km values. The low efficiency of these compounds as electron donors is apparently not due to their weak binding to QCR. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is very specific and differs from that in ubiquinol-cytochrome c reductase.