Tomato cDNA and genomic clones were isolated by using as a probe a cDNA clone that had originally been identified by its ability to direct the synthesis of a biotin-containing polypeptide in Escherichia coli. The nucleotide sequences of the newly isolated cDNAs indicate that they are clones of a single mRNA molecule. However, one of the cDNA clones contains an insertion of a sequence which we identified as an unspliced intron. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed similarity to regions of previously sequenced biotin enzymes, indicating that the isolated cDNAs code for a biotin-containing protein. Portions of the cDNAs were expressed in E. coli as glutathione S-transferase or beta-galactosidase fusion proteins. Each fusion protein was purified and used to immunize rabbits. The resulting antisera recognized a 78-kDa biotin-containing polypeptide in tomato leaf extracts. In addition, both antisera specifically inhibited beta-methylcrotonyl-CoA carboxylase activity in extracts from tomato leaves. These characterizations have identified the isolated tomato cDNAs and genes as coding for the 78-kDa biotin subunit of beta-methylcrotonyl-CoA carboxylase. Comparison of the deduced amino acid sequence of the biotin subunit of beta-methylcrotonyl-CoA carboxylase with other biotin enzymes suggest that this subunit contains the biotin carboxylase and biotin carboxyl-carrier domains.