A new serine protease inhibitor, designated cvSI-2, was purified and characterized from the plasma of the eastern oyster, Crassostrea virginica. CvSI-2 inhibited the serine protease subtilisin A in a slow-tight binding manner, with an overall dissociation constant Ki* of 0.18 nM. It also inhibited perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus. Sequencing of cvSI-2 cloned cDNA revealed an open reading frame of 258 bp encoding a polypeptide of 85 amino acids, with the 18 N-terminal amino acids forming a signal peptide. The mature cvSI-2 molecule predicted consisted of 67 amino acids with 12 cysteine residues and a calculated molecular mass of 7202.96 Da. Overall 91% of the cvSI-2 amino acid sequence predicted from cDNA was confirmed by tandem mass spectrometry sequencing of purified cvSI-2. In addition, serine 43 and a threonine substitution at this position were observed. CvSI-2 amino acid sequence showed a 38% identity and 54% similarity with that of cvSI-1, the first protease inhibitor purified and characterized from a bivalve mollusc. Like cvSI-1, cvSI-2 gene was expressed in the basophil cells of digestive tubules. BLAST search found multiple ESTs from the eastern oyster, Pacific oyster, Mediterranean mussel, and sea vase, a tunicate, which could encode proteins with sequences similar to cvSI-1 and cvSI-2. Our findings indicate that cvSI-1 and cvSI-2 are members of a novel family of serine protease inhibitors in bivalve molluscs and perhaps other marine invertebrates, which share the characteristic cysteine array C-X(4-9)-C-X(4-6)-C-X(7)-C-X(4)-C-T-C-X(6-9)-C-X(5)-C-X(3-7)-C-X(6-10)-C-X(4)-C-X-C.