5-Iodo-2'-deoxyuridine (IUDR) is a potent topical antiviral agent in experimental animals but is less active in man for treating cutaneous viral infections. We have shown here that IUDR is 5 times less active in human keratinocytes infected in vitro with herpes simplex virus type 1 than in guinea pig embryo cells infected in culture. To account, in part, for this difference in activity of IUDR, we measured the capacity of these different cultures to catabolize and thus inactivate the drug. IUDR is catabolized by thymidine phosphorylase; activity of this enzyme was very high in human keratinocytes in vitro but was very low in guinea pig embryo cells. The antiviral activity of IUDR in human keratinocytes, however, was not increased by inhibiting thymidine phosphorylase; inhibiting thymidine phosphorylase apparently increased the availability of thymidine that would compete with IUDR and, indeed, the activity of IUDR in infected cells was reduced by addition of thymidine to the medium. These data indicate that the catabolism of IUDR and related analogs alters antiviral activity in human keratinocytes.