Herpes simplex virus (HSV) ocular virulence has been associated with strain sensitivity to mouse interferon (IFN)-alpha/beta. To identify the region of the virus genome associated with heightened resistance to this cytokine, intertypic recombinants were constructed using the intact genome of avirulent, IFN-sensitive HSV type 1 (strain 35) and XbaI-digested DNA from virulent, IFN-resistant HSV type 2 (strain 186). An intertypic recombinant, designated HSV-R4, was isolated which grew to titres 10- to 100-fold higher than HSV-1(35) in mouse ocular tissue in vivo, and induced stromal keratitis. The recombinant which was several orders of magnitude more resistant to mouse IFN-alpha/beta than HSV-1(35) had a genome composed of HSV-1(35) DNA except for a 12 kb fragment (0.15 to 0.23 map units) derived from HSV-2(186). To define the IFN resistance locus further, three overlapping subclones of this 12 kb fragment were constructed from the HSV-2(186) genome and subjected to marker rescue experiments. The cloned BamHI D fragment was the only subclone that promoted HSV-1(35) ocular growth in vivo. An intertypic recombinant, designated HSV-R(BD), was isolated from the 35 x 186 BamHI D transfection progeny pool. This recombinant, in contrast to HSV-1(35), was several orders of magnitude more resistant to mouse IFN-alpha/beta inhibition in vitro, grew 10- to 100-fold better in mouse ocular tissue in vivo, and caused severe necrotizing stromal keratitis in BALB/c mice. Analysis of the recombinant genome indicated that the HSV-2 genetic information responsible for IFN resistance of HSV-R(BD) was located within the BamHI D fragment, most likely mapping to that region containing three partial open reading frames designated UL14, UL15 and UL16. The products encoded by this region remain to be identified.