The wild-type ICP4s (infected-cell polypeptide 4) encoded by herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are functionally interchangeable. In order to test the functional interchangeability of their intramolecular domains, a series of intertypic ICP4 genes was constructed and characterized to determine if any of the encoded chimeric proteins were functionally impaired. We generated the recombinants in Escherichia coli using cloned ICP4 genes and the lambda recombination vectors developed by D. Carroll and R. S. Ajioka (1980, Gene 10, 273-281) and D. Carroll, R. S. Ajioka, and C. Georgopoulos (1980, Gene 10, 261-271). We chose to generate the recombinants in E. coli in order to avoid imposing any restrictions with respect to the biological activities of their chimeric protein products. Six different recombinants encoding chimeric ICP4s were studied. As determined by restriction enzyme analysis, one of the six encodes an ICP4 protein whose amino-terminus is type 1 and whose carboxy-terminus is type 2. Five recombinants encode ICP4 proteins whose amino-termini are type 2 and carboxy-termini, type 1. The recombinant ICP4 proteins were assessed for their ability to stimulate transcription driven by the HSV-1 thymidine kinase promoter and for their ability to complement the growth of d120 and hr259, deletion mutants in HSV-1 and HSV-2 ICP4, respectively. All six recombinants exhibited wild-type levels of functional activity in both assay systems, demonstrating the colinearity of sequences specifying the intramolecular domains of HSV-1 and HSV-2 ICP4 and their functional interchangeability.