Tick serine proteases are involved in blood digestion as well as complement activation and other immune processes, making them viable anti-tick vaccine candidates. We detected serine protease activity in midgut and salivary gland homogenates and saliva of black-legged ticks, Ixodes scapularis. Inhibiting proteolytic activity using N-á-p- tosyl-L-chloromethyl ketone (TLCK), antipain, aprotinin, and benzamidine confirmed the presence of serine proteases in this tick. cDNA coding for a serine protease was cloned and sequenced from a cDNA library prepared from adult I. scapularis salivary glands. Expression analysis by semi-quantitative RT-PCR revealed that the I. scapularis serine protease was predominantly expressed in midgut and hemolymph when compared to salivary gland, and expression was up-regulated during the feeding process. In vitro studies suggest that this protease is involved in blood meal digestion. RNAi was used in vivo to evaluate the functional role of this protease on digestion. The full length cDNA serine protease gene was cloned into a mammalian expression vector for recombinant protein expression. Expression, purification, and the functional characterization of the recombinant protein will be discussed.