An assay for 6-thioguanine (6-TG) nucleotide, a major metabolite of the cytotoxic drugs azathioprine, 6-mercaptopurine and 6-thioguanine in human red blood cells (RBCs) has been developed. The metabolite was not detected in RBCs when azathioprine or 6-mercaptopurine was incubated with whole blood in-vitro. The assay for intracellular 6-TG nucleotide is specific and requires 8 times 108 RBCs (100 μl packed cells) for which the limit of sensitivity is 30 pmole 6-TG nucleotide. Pre-dose blood samples were obtained, 12 h after the last azathioprine dose, from 10 renal transplant recipients with stable functioning cadaver grafts on a total daily dose of 150 mg azathioprine. The mean 6-TG nucleotide concentration was 171 pmole/8 times 108 RBCs(s.d. = 84). The assay is also suitable for use in measuring 6-TG nucleotide in the RBCs of leukaemic children undergoing 6-mercaptopurine treatment.