A series of new 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine and uridine compounds (11, 16) was synthesized by the regiospecific addition of HOI to the vinyl substituent of 5-vinyl-2'-deoxyuridine (10a), 5-vinyl-2'-fluoro-2'-deoxyuridine (10b), 5-vinyluridine (10c), and (E)-5-(2-iodovinyl)-2'-deoxyuridine (4b). Treatment of the iodohydrins 11a-c with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-iodoethyl) derivatives (12a-c). In contrast, reaction of 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine (11a) with sodium carbonate in methanol afforded a mixture of 5-(1-hydroxy-2-methoxyethyl)-2'-deoxyuridine (13) and 2,3-dihydro-3-hydroxy-5-(2'-deoxy-beta-D-ribofuranosyl)- furano[2,3-d]pyrimidin-6(5H)-one (14). The most active compound, 5-(1-methoxy-2-iodoethyl)-2'-deoxyuridine (12a, ID50 = 0.1 micrograms/mL), which exhibited antiviral activity (HSV-1) 100-fold higher than that of the 5-(1-hydroxy-2-iodoethyl) analogue (11a), was less active than IVDU or acyclovir (ID50 = 0.01-0.1 micrograms/mL range). The C-5 substituent in the 2'-deoxyuridine series was a determinant of cytotoxic activity, as determined in the in vitro L1210 screen, where the relative activity order was CH(OH)CHI2 (16) greater than CH(OMe)CH2I (12a) greater than CH(OH)CH2I (11a) congruent to CH(OH)CH2OMe (13). The 2'-substituent was also a determinant of cytotoxic activity in the 5-(1-hydroxy-2-iodoethyl) (11a-c) and 5-(1-methoxy-2-iodoethyl) series of compounds, where the relative activity profile was 2'-deoxyuridine greater than 2'-fluoro-2'-deoxyuridine greater than uridine (11a greater than 11b greater than or equal to 11c; 12a greater than 12b greater than 12c). The most active cytotoxic agent (16), possessing a 5-(1-hydroxy-2,2-diiodoethyl) substituent (ED50 = 0.77 micrograms/mL), exhibited an activity approaching that of melphalan (ED50 = 0.15 micrograms/mL). All compounds tested, except for 13 and 14, exhibited high affinity (Ki = 0.035-0.22 mM range relative to deoxyuridine, Ki = 0.125) for the murine NBMPR-sensitive erythrocyte nucleoside transport system, suggesting that these iodohydrins are good permeants of cell membranes.