The natural products epothilone and bleomycin are assembled by hybrid polyketide/nonribosomal peptide synthetases. Of note in these assembly lines is the conversion of internal cysteine residues into thiazolines and their subsequent oxidation to heteroaromatic thiazole rings. We have excised the EpoB oxidase domain, EpoB-Ox, proposed to be responsible for thiazoline to thiazole oxidation in epothilone biosynthesis, and expressed it in soluble form in Escherichia coli. The purified domain is an FMN-containing flavoprotein that demonstrates thiazoline to thiazole oxidase activity when incubated with thioester substrate mimics. Kinetic parameters were determined for both thiazoline and oxazoline substrates, with k(cat) values ranging between 48.8 and 0.55 min(-1). While the physiological electron acceptor is not yet known, molecular oxygen is needed in these in vitro assays to mediate reoxidation of reduced FMN. Additionally, the oxidase domain-containing BlmIII from the bleomycin assembly line was heterologously expressed and purified. BlmIII is also an FMN-containing protein with activity similar to EpoB-Ox. This work marks the first direct characterization of nonribosomal peptide synthetase oxidase domain activity and will lead to further exploration of these flavoproteins.