Here we describe the recombinant production and purification of a novel anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N), in Escherichia coli. Initial attempts to express CV-N using a vector containing an ompA signal peptide sequence resulted in production of an intractable mixture of the full-length (101 amino acid residue) protein and a truncated form lacking the first two N-terminal amino acids. The truncated protein was observed regardless of the host cell line, culture conditions, or induction time. These observations suggested that an as yet unidentified protease or peptidase was responsible for proteolytic cleavage between the second and third N-terminal amino acids of CV-N when presented as an ompA-CV-N fusion protein. When the ompA signal peptide sequence was replaced by a pelB signal peptide sequence, CV-N was produced in high yield as a single, homogeneous protein. This was confirmed by electrospray ionization mass spectrometry and N-terminal sequencing. This expression system provides a basis for large-scale production of clinical grade CV-N for further research and development as an anti-HIV microbicide.