The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4-2OH, N5-2OH, and N7-2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5-2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(-). N5-2OH was the least toxic (IC50, 43-70 μM), and N7 and N7-2OH were the most toxic (IC50, 18-49 μM). The highest boron uptake was seen with N7-2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5-2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5-2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 ± 2.3 and 2.2 μg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5-2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(-) tumors were 39.8 ± 10.8 and 12.4 ± 1.6 μg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 μg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5-2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.