The emergence of cross-resistance to various antiviral drugs was investigated both in vivo and in vitro for herpes simplex virus type 1 (HSV1) resistant to idoxuridine IUdR 0.24%) obtained by seven successive passages from P0 to P7) in rabbit keratitis treated by IUdR. The viral population obtained at the seventh IUdR passage P7) showed an activity of the thymidine kinase (TK) reduced to 5.6% of the parental strain (PO); moreover, most of the clones of P7 showed an altered TK phenotype determined by the [l25I]iododeoxycytidine (IDC) procedure. In rabbit keratitis, IUdR-resistant viral population P7 showed cross-resistance to bromovinyl desoxyuridine (BVDU) (0.5%) and to acyclovir (ACV) (3%). Under trifluorothymidine (1%) treatment, P7 showed an intermediate sensitivity. HSV1 at P7 remained sensitive to adenine arabinoside (Ara A) (3%) and to dihydroxy-propoxymethylguanine used at high concentration (3%). The in vitro sensitivity determination to various antiviral drugs was investigated by dye-uptake assay for the initial viral population PO and for HSV1 collected under IUdR treatment at the third (P3) and the seventh (P7) passages. Cross-resistance to TK-dependent drugs, such as IDC, BVDU, and ACV were found at P7. P7 remained sensitive to Ara A and to phosphonoformic acids antiviral drugs known not to be dependent on viral TK.