Administration of nucleotide mixtures has been shown to restore and sustain the proliferation of leukocytes and enterocytes. Since it has been suggested that cancer cells use exogenous nucleotides more efficiently than normal cells, we hypothesized that administration of nucleotide mixtures would also stimulate the proliferation of cancer cells, thereby increasing the number of cells targeted by the thymidine analog 5-[(125)I]iodo-2'-deoxyuridine ([(125)I]IUdR). We first evaluated the influence of different deoxyribonucleoside mixtures on the DNA incorporation of [(125)I]IUdR in 3 human glioblastoma cell lines. Results showed that a 4-h coincubation with a mixture of identical concentration (10 microM) of deoxyadenosine, deoxyuridine, deoxyguanosine and deoxycytidine (AUGC) increased by 8.5-, 6.2-, and 2.0-fold the rate of DNA incorporation of [(125)I]IUdR in exponentially growing LN229, U87 and U251 cells, respectively. Replacing deoxyuridine by thymidine (ATGC) reversed the effect of the mixture, whereas removing deoxyuridine allowed a mixture of 10 microM AGC to increase by 2.2-fold the rate of DNA incorporation of [(125)I]IUdR in LN229 cells. Furthermore, the rate of DNA incorporation of [(125)I]IUdR in LN229 and U87 cells was increased up to 19.9- and 9.4-fold, respectively, by extending the coincubation time with 10 microM AUGC to 9 h, and up to 40.9- and 26.8-fold by incubating confluent cells for 4 h with 10 microM AUGC. Flow cytometry analysis showed that exposure of confluent cells to AUGC increased the percentage of cells in S phase of the cell cycle. Thus, co-administration of a balanced deoxyribonucleoside mixture may improve the use of radiolabeled nucleotide analogs, such as [(125)I]IUdR, for the targeting of cancer cells.