Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K(I) (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.