Metabolism of thiopurine drugs--azathioprine, 6-mercaptopurine, and 6-thioguanine--has provided a powerful pharmacogenetic model incorporating polymorphism of the enzyme thiopurine methyltransferase (TPMT) and the primary active metabolite, thioguanine nucleotide (TGN). However, a sense of uncertainty about the usefulness of TGNs and other thiopurine metabolites has appeared. This review critically appraises the basis of thiopurine metabolism and reveals the problems and complexities in TGN research. Erythrocyte TGN is used in transplantation medicine and in chronic inflammatory conditions such as Crohn's disease, as a "surrogate" pharmacokinetic parameter for TGN in the target cells: leukocytes or bone marrow. It is not generally appreciated that erythrocytes do not express the enzyme IMP dehydrogenase and cannot convert mercaptopurine to TGN, which explains some of the confusion in interpretation of erythrocyte TGN measurements. TGN routinely measured in erythrocytes derives from hepatic metabolism. Another concern is that TGN are not generally assayed directly: most methods assay the thiopurine bases. Ion-exchange HPLC and enzymatic conversion of TGNs to nucleosides have been used to overcome this, and may reveal undisclosed roles for an unusual cytotoxic nucleotide, thio-inosine triphosphate, and methylated thiopurines. There appear to be additional interactions between xanthine oxidase and TPMT, and folate and TPMT, that could predict leukopenia. Difficult questions remain to be answered, which may be assisted by technological advances. Prospective TGN studies, long overdue, are at last revealing clearer results.