Some biological conditions of the focal necrotic hepatitis test for the differentiation between herpes simplex virus (HSV) types 1 and 2 were investigated. Most of 13 different strains of mice tested were found usable in the test. An upper age limit (4 weeks) for the appearance of focal necrotic liver lesions was found in one strain of mice, while this was not seen in another strain. The minimum dose in 3- to 4-week-old mice was found to be as small as 102 to 103 p.f.u. in 0.1 ml of diluent. Suckling rats and hamsters, aged up to 7 and 14 days, respectively, were found to be convenient as alternative test animals. Finally, it was observed that focal necrotic hepatitis did not develop in the nude mouse with thymic aplasia on intraperitoneal inoculation of HSV type 2. The possible involvement of the thymus in the pathogenesis of the focal necrotic lesions is briefly discussed.