Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2'-deoxyadenosine were 23 and 16 microM, respectively. Purine riboside was a competitive inhibitor with Ki of 5 microM, whereas 2'-3'-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 microM). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg2+ (100 microM) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, alpha-adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3'-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2'-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.