High-pressure differential pulse voltammetry and cyclic voltammetry were employed to determine the reaction volume associated with electron transfer in cytochrome c and a series of ruthenium-modified cytochromes c. The reduction of Cyt c(III), either in the native or ruthenium-modified form, is characterized by a reaction volume of -14.0 +/- 0.5 cm(3) mol(-1) when measured versus a Ag/AgCl, KCl(sat’d) reference electrode. A detailed study of the reference electrode system resulted in a value of -9.0 +/- 0.6 cm(3) mol(-1) for the contribution to Delta V for the net reaction Cyt c(III) + Ag(s) + Cl-(aq) --> Cyt c(II) + AgCl(s) from the reference electrode components Ag(s), Cl-(aq), and AgCl(s). It follows that the absolute molar volume of Cyt c(III) exceeds that of Cyt c(II) by only 5.0 +/- 0.8 cm(3) mol(-1) (mu = 0.1 M, pH = 7), i.e. much less than the value of 24 cm(3) mol(-1) reported in the recent literature. Reaction volumes for a series of intramolecular electron-transfer reactions of the type trans-(NH3)(4)Ru-III(L)-Cyt c(II) --> trans-(NH3)(4)R(II)(L)-Cyt c(III) were found to be 31.7 +/- 1.2 (L = NH3), 21.1 +/- 1.0 (L = isonicotinamide), 23.3 +/- 0.6 (L = pyridine), and 18.6 +/- 0.4 cm(3) mol(-1) (L = 3,5-lutidine). This volume increase is mainly assigned to a decrease in electrostriction during the reduction of the ruthenium center and can be correlated with the number of coordinated ammine ligands. It is concluded that cytochrome c undergoes only a small volume change during electron-transfer reactions.