We investigated the potential of radiolabelled 5-iodo-2'-deoxyuridine (IUdR) as a pharmacodynamic probe for use with positron emission tomography (PET) in studies of early proliferative response to anticancer treatment. Using the hormone-responsive rat mammary carcinoma OES.HR1, we used a multiple radiotracer method to examine treatment-induced changes in 24 h tumour retention of [131I]IUdR, uptake of [3H]2-deoxy-D-glucose ([3H]DG) together with [99mTc]hexylmethylpropylene amineoxine ([99mTc]HMPAO) uptake as a measure of blood flow. Radiotracer data were compared with macroscopic changes in tumour growth, and cell proliferation as determined by DNA histogram flow cytometry. From 4 days after tumour growth arrest induced by oestrogen ablation, a sustained fall in tumour cell proliferation was demonstrated, which was associated with reduced tumour uptake of each tracer. Whereas reduced levels of tumour [3H]DG could be accounted for by changes in blood flow, this was not the case for [131I]IUdR, which was found to be closely related to percentage S-phase cells within tumour (r = 0.73, p < 0.002). It was also estimated that residual levels of radioiodide may contribute significantly, to the low levels of retained radioactivity associated with responding tumours at 24 h following IUdR administration, suggesting that metabolite correction methods should be implemented as part of IUdR PET imaging protocols. We conclude that [124I]IUdR is a promising alternative to [18F]fluorodeoxyglucose ([18F]FDG) for the early assessment by PET of tumour response to treatments directed at targets associated with cell proliferation.