Summary: Three classes of cytochrome a deficient mutants of Bacillus subtilis have been isolated. These classes were defined by concentration ratios of cytochromes a, b and c relative to the parent strain, and by responses to non-fermentable substrates. The cytochrome deficiencies were not due to blocks in haem biosynthesis or to failure of the control mechanisms which alter the respiratory chain during growth of B. subtilis. Mutational loss of cytochrome a did not reduce proportionately the rate of oxygen consumption by late exponential phase cells, although exponential growth rates were generally lower than the parent strain. The cytochrome system of wild-type B. subtilis appears to contain cytochromes a (or a+a3), b, c, c1, and o. A previously unreported absorption maximum at 617 nm was discovered, which was elevated in some of the cytochrome a deficient mutants. In these mutants, a new maximum also appeared at 627 nm.