The heterogeneity of beef heart cytochrome c, obtained in the native, monomeric form by gel filtration of Sephadex G-75, has been studied, and the following results were obtained: (1) By means of disc electrophoresis on polyacrylamide gel cytochrome c, extracted by dilute sulphuric acid at pH 4.0, was separated into four subtractions (Cy I 890%, Cy II 9.7%, Cy III 1.1%, and Cy IV 0.2%). A heterogeneity was also demonstrated by chromatography on Duolite CS-101, but five com- ponents were then consistently revealed. The subtractions separated by electrophoresis and chromatography were shown to be compatible with each other except that two of the chromatographic fractions demonstrated almost the same mobility on disc electrophoresis. (2) Almost the same percentage distribution of subtractions was obtained whether cytochrome c was extracted by dilute sulphuric acid or by distilled water/saline- buffer. Prolonged autolysis of the minced muscle before extraction, however, resulted in a higher percentage of the minor subtractions (Cy II [long dash] Cy IV), but no conclusive evidence for interconvertibility was obtained. (3) The distinctions so far observed between the various electro-phoretic and chromatographic subtractions of cytochrome c is a difference in net charge; the main fraction (Cy I) being the most positively charged at neutral pH. Detailed chemical and physical properties are not yet established.