The plasma concentrations and tissue distribution of thiopurines were studied in mice after oral administration of 50 mg/kg azathioprine (AZA) using HPLC analysis. Peak concentrations of AZA and three other thiopurine metabolites in plasma [thiouric acid (TUA) greater than 6-mercaptopurine (6-MP) greater than AZA greater than 8-hydroxy-AZA] were observed as early as 10 min after drug application, thus indicating fast absorption and extensive metabolism of AZA, and were followed by a rapid decline. The extraction of thiopurines from organs (intestinal mucosa, liver, kidney, testes, spleen, and bone marrow) and from red blood cells (RBCs) was preceded by an acid hydrolysis procedure resulting in the release of thiopurine bases from their corresponding ribonucleotides. 6-MP, 6-thioxanthene (6-TX), 6-thioguanine (6-TG), TUA, and 8-hydroxy-6-MP (8-OH-6-MP) were extracted from the organs, whereas only 6-MP and 8-OH-6-MP were found in the processed RBCs. Initially, high concentrations of TUA, the endpoint of metabolic AZA degradation, were detected in the intestinal mucosa and in the liver. This provides evidence for a first-pass metabolism of AZA in these two organs. The initial concentrations of 6-MP extracted from the organs were about 10-fold those found in plasma. This indicates rapid cellular uptake of 6-MP and an accumulation of 6-MP derivatives that can be explained by formation of the 6-MP ribonucleotide thioinosine monophosphate (TIMP). With the exception of plasma and RBCs, 6-TG, which may originate from intracellular 6-thioguanosine nucleotides (TGNs), was extracted from all organs examined in the study. From the sequence of appearance of 6-MP, 6-TX, and 6-TG extracted from spleen and bone marrow homogenates, it can be assumed that formation of TGN occurs via the nucleotide interconversion pathway TIMP----6-thioxanthosine monophosphate----6-thioguanosine monophosphate. The highest concentrations of 6-TG derivatives were found in the spleen and bone marrow. This correlates with the clinical and experimental observation that AZA cytotoxicity mainly affects bone-marrow stem cells and lymphocytes and supports the hypothesis (derived from in vitro experiments) that the incorporation of TGN into DNA is the cytotoxic mechanism of AZA and 6-MP.