The aim of this study was to determine the most suitable iodonucleoside analogs for use in tissue proliferation imaging by means of single photon emission tomography (SPECT). In this study, 5-[(125)I]iodo-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)uracil ([(125)I]FITAU, 1E) and 5-[(125)I]iodo-1-methyl-(2-deoxy-2-bromo-beta-D-arabinofuranosyl)uracil ([(125)I]IMBAU, 1F) were synthesized and their biological data were compared with previously published results regarding 4'-thio nucleoside analogs and the reference compound 5-[(125)I]iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil ([(125)I]FIAU, 1D). 5-Iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (FIAU, 2D), 5-iodo-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)uracil (FITAU, 2E), and 5-iodo-1-methyl-(2-deoxy-2-bromo-beta-D-arabinofuranosyl)uracil (IMBAU, 2F) were successfully labeled with (125)I and their in vitro cytosolic thymidine kinase (TK(1)) phosphorylation, recombinant thymidine phosphorylase enzymatic catabolism, TK(1)-dependent cell uptake, and in vivo biodistribution in normal mice were evaluated. Five compounds (1B, 1C, 1D, 1E, and 1F) were stable against C-N glycoside degradation induced by recombinant thymidine phosphorylase. However, 5-[(125)I]iodo-2'-deoxyuridine ([(125)I]IUdR, 1A) was not shown to be stable against such degradation. The TK(1) assay showed that [(125)I]FIAU (1D) expressed 16% of the phosphorylation potential of [(125)I]IUdR (1A). Furthermore, [(125)I]FITAU (1E) was shown to have reduced phosphorylation potential, in comparison with that of [(125)I]IUdR (1A) (<0.01). [(125)I]IMBAU (1F) did not show any phosphorylation. In vitro cell uptake and in vivo proliferation-selective uptake of each nucleoside was largely dependent on its potential as a TK(1) substrate. Neither [(125)I]FITAU (1E) nor [(125)I]IMBAU (1F) were shown to have distinct TK(1)-dependent cell uptake and retention in the proliferating tissues. From these results, we concluded that [(125)I]FITAU (1E) and [(125)I]IMBAU (1F) are not effective as imaging agents of cell proliferation. The biological data obtained with these nucleosides were compared, and requirements for the design of pharmaceutically useful radioiodinated nucleoside analogs were also considered.