We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis arietans, that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn(2+)-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA(2) (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis arietans. The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.