Ornamental tobacco (Nicotiana langsdorffii X N. sanderae) secretes a limited array of proteins (nectarins) into its floral nectar. Careful sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of tobacco nectar revealed that a broad protein band from 61 to 65 kD actually consists of five discrete protein bands. N-terminal sequencing and tryptic peptide mass spectrometry fingerprint analysis demonstrated that the upper three bands are isoforms of the same protein, NEC5 (Nectarin V), whereas the lower two bands, NEC4 (Nectarin IV), are related to each other but not to NEC5. Reverse transcription-polymerase chain reaction (RT-PCR) based upon N-terminal sequence of NEC5 generated a short cDNA that encoded the N terminus of the NEC5 protein. Two rounds of inverse-PCR using genomic DNA permitted the isolation of approximately one-half of the coding region of the nec5 gene along with 787 nucleotides of the 5′-flanking region. This DNA fragment was used as a probe to isolate a near full-length nec5 clone from a nectary-derived cDNA library. BLAST analysis identified the nec5 cDNA as a berberine bridge enzyme-like protein. Approximately 40% of the cDNA sequence corresponded to peptides that were identified by tryptic peptide mass spectrometry fingerprint analysis of the NEC5 protein, thereby confirming that this cDNA encoded the NEC5 protein. In-gel assays also demonstrated that NEC5 contains a covalently linked flavin, and it possesses glucose oxidase activity. RT-PCR-based expression analyses showed that nec5 expression is limited exclusively to the nectary gland during late stages of floral development.