Biotin-containing enzymes catalyze critical reactions in diverse metabolic processes. In contrast to the extensive characterizations of biotin-containing enzymes from animal and microbial sources, little is known about these enzymes from plants. I have isolated and sequenced cDNA clones coding for the biotin-containing subunit [beta]-methylcrotonyl-CoA carboxylase (MCCase) from tomato, the first molecular cloning of this enzyme from any organism, and the first molecular cloning of a plant biotin-containing enzyme. Comparison of the deduced amino acid sequence from these clones with other biotin-containing enzymes suggests that the 78 kD subunit carries biotin carboxylase and biotin carboxyl carrier functions. I have shown that this MCCase has an apparent molecular weight of about 1,200 kD and is composed of two types of subunits, 78 kD (the biotin-containing subunit) and 60 kD (the biotin-free subunit). I have shown that MCCase is distributed among all organs examined from tomato plants. MCCase activity is highest in non-photosynthetic tissues, especially roots. The biochemical mechanism by which the difference in specific activity of MCCase among leaves and roots was investigated. These studies indicated that MCCase activity is regulated by the differential biotinylation of the enzyme.