Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [C-14] ADP-ribose uptake values. When [adenosine-C-14] NAD was used instead of free [C-14] ADPribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-C-14] NAD by Cibacron Blue. ADPribose uptake in erythrocytes appeared to be complex: upon incubation with free [C-14] ADPribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [C-14] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [C-14] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-C-14] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADPribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.