The diffusion coefficient and the effective charge number of cytochrome c as a function of ionic strength, temperature and pH have been measured. The measurements were carried out using a method based on a convective diffusion process across a porous membrane. The effect of ionic strength was studied in an NaCl solution the concentration of which varied from 0.001 to 1.0 M. The temperature range studied was 10-50 degrees C, and the pH values studied were 4.0, 6.5 and 8.25. The diffusion coefficient is fairly constant as a function of ionic strength and pH, and Walden's rule is valid in the temperature range studied. The effective charge number is practically constant (ca. 2) in the concentration range studied, except in 0.001 M solution, where it is the same as the titrated value. The charge number decreases slightly in the temperature range 10-30 degrees C, but seems to drop suddenly to zero at ca. 40 degrees C. Measurements using heavy water (D2O) as a solvent instead of water did not give zero charge at 40 degrees C for cytochrome c.