Pharmacokinetics and metabolite analysis of radioiodinated 5-iodo-4'-thio-2'-deoxyuridine (ITdU), a potential tumor cell proliferation-imaging agent, were investigated in rodents. METHODS Blood and urine metabolites of radiolabeled ITdU were analyzed by thin-layer chromatography in rats. Tissue fractionation studies in mice were also performed to investigate the incorporation of (125)I-ITdU into DNA. RESULTS The blood distribution study suggested the influx of (125)I-ITdU into rat peripheral blood cells. The blood metabolism of (125)I-ITdU in vitro was negligible. Plasma analysis at 60 min after injection showed that 50% of the radioactivity was intact (125)I-ITdU. The plasma clearance of (125)I-ITdU showed a biexponential function with half-lives of 0.2 and 29.5 min, representing 94.2% and 5.8% of plasma (125)I-ITdU concentration, respectively. (125)I-ITdU was excreted in urine 86% intact at 3.5 h after injection. Extraction of the tissues demonstrated that >70% of the activity in the duodenum, spleen, and thymus was incorporated into DNA at 8 h after injection. On the other hand, <8% of the activity in the liver was in the DNA fraction at that time. CONCLUSIONS These data demonstrate that (125)I-ITdU is less susceptible against catabolism and readily incorporated into DNA in proliferating tissues. Therefore, (125)I-ITdU may be useful in measuring DNA synthesis with SPECT.