The biotin enzyme, 3-methylcrotonyl-CoA carboxylase (MCCase) (3-methylcrotonyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.4), catalyzes a pivotal reaction required for both leucine catabolism and isoprenoid metabolism. MCCase is a heteromeric enzyme composed of biotin-containing (MCC-A) and non-biotin-containing (MCC-B) subunits. Although the sequence of the MCC-A subunit was previously determined, the primary structure of the MCC-B subunit is unknown. Based upon sequences of biotin enzymes that use substrates structurally related to 3-methylcrotonyl-CoA, we isolated the MCC-B cDNA and gene ofArabidopsis. Antibodies directed against the bacterially produced recombinant protein encoded by the MCC-B cDNA react solely with the MCC-B subunit of the purified MCCase and inhibit MCCase activity. The primary structure of the MCC-B subunit shows the highest similarity to carboxyltransferase domains of biotin enzymes that use methyl-branched thiol esters as substrate or products. The single copy MCC-B gene of Arabidopsis is interrupted by nine introns. MCC-A and MCC-BmRNAs accumulate in all cell types and organs, with the highest accumulation occurring in rapidly growing and metabolically active tissues. In addition, these two mRNAs accumulate coordinately in an approximately equal molar ratio, and they each account for between 0.01 and 0.1 mol % of cellular mRNA. The sequence of theArabidopsis MCC-B gene has enabled the identification of animal paralogous MCC-B cDNAs and genes, which may have an impact on the molecular understanding of the lethal inherited metabolic disorder methylcrotonylglyciuria.