A series of hexofuranosyladenine nucleosides have been tested as substrates and inhibitors of adenosine deaminase from calf intestinal mucosa. The nucleosides differed from each other in configuration at the various carbon atoms of the hexose and had either a methyl group or hydroxymethyl group at the terminal position. It has been confirmed that the best substrates have the beta-D or alpha-L configuration at the anomeric position and an hydroxyl group on the same side of the furanose ring as adenine. However, these properties are not minimal and other nucleosides will act as substrates even if they do not have the preferred configurations or groups available. The effect of having two hydroxyl groups in the same region of the molecule and in the preferred configurations was to greatly reduce Vmax. Most structural changes resulted in changes in Vmax, whereas KM values remained fairly close. Only a change in configuration of the hydroxyl group at C-5' caused a dramatic change in affinity, as reflected in the KM. All nucleosides exhibited competitive inhibitory kinetics. In the latter studies also, a change of configuration at C-5' greatly affected binding.