<jats:title>ABSTRACT</jats:title> <jats:p> The coenzyme B <jats:sub>12</jats:sub> -dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of <jats:italic>n</jats:italic> -butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of <jats:italic>n</jats:italic> - and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer <jats:italic>Streptomyces cinnamonensis</jats:italic> . Mutants prepared by inserting a hygromycin resistance gene ( <jats:italic>hygB</jats:italic> ) into either <jats:italic>icmA</jats:italic> or <jats:italic>mutB</jats:italic> , encoding the large subunits of ICM and MCM, respectively, have been characterized. The <jats:italic>icmA</jats:italic> :: <jats:italic>hygB</jats:italic> mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The <jats:italic>mutB</jats:italic> :: <jats:italic>hygB</jats:italic> mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. <jats:sup>13</jats:sup> C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, <jats:italic>n</jats:italic> -butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.