Here we describe a cDNA encoding the second asparaginyl endopeptidase/legumain (HlLgm2) from the midgut of the ixodid tick Haemaphysalis longicornis. Endogenous HlLgm2 was expressed in all the developmental stages of the tick, localized mainly in the midgut epithelium and was up-regulated by the host blood-feeding process, as demonstrated by immunoblotting and immunohistochemistry. RT-PCR and real-time PCR showed that the HlLgm2 gene was expressed at a lower level during all phases of blood-feeding than our previously characterized legumain (HlLgm) gene from the same tick. More strikingly, there was no expression of HlLgm2 mRNA beyond 96 h of blood-feeding, while HlLgm mRNA expression continued until full engorgement. Escherichia coli-expressed recombinant HlLgm2 (rHlLgm2) efficiently hydrolysed the legumain-specific synthetic substrate. rHlLgm2 activity was inhibited by iodoacetamide and N-ethylmaleimide and also by Fe(2+), Cu(2+), Co(2+) and Ni(2+). rHlLgm2 digested bovine haemoglobin and exhibited strict specificity for the asparaginyl bonds on the carboxy-terminal side of a peptide, as demonstrated by internal amino acid sequence analysis of the cleaved bovine serum albumin products. Our results suggest that HlLgm2, together with HlLgm, plays a pivotal role in host blood-meal digestion process.