Marmoset herpesvirus (MarHV) deletion mutants in the thymidine kinase (TK) gene were isolated after infection of OMK cells with TK+ MarHV DNA and the hybrid plasmid, pMAR401, which lacks a 2.6-kb KpnI-M fragment in the coding region of the MarHV TK gene. After plaque purification in TK- HeLa(BU25) cells, the DNA's from five recombinant araT-resistant MarHV clones were analyzed with restriction nucleases to verify that the 2.6-kb KpnI-M fragment (and a 0.9-kb BglII-Q fragment) were deleted from the viral DNA's. Molecular hybridization experiments using 32P-labeled pMAR4 probes and viral DNA fragments also showed that the recombinant viral DNA's lacked the KpnI-M and BglII-Q fragments, that BamHI-I of parental virus was shortened, and that three new HindIII fragments replaced the parental virus HindIII-G fragment. The recombinants did not induce TK activity in LM (TK-) cells. To study the relative virulence of the recombinants, 3-week-old Swiss mice were injected intracerebrally (Ic) or subcutaneously (Sc) in the sacro-lumbar region with either parental or recombinant viruses. The LD50 for the parental virus was 3 p.f.u. (Ic) and 7,600 p.f.u. (Sc). The recombinant viruses were significantly less virulent than TK+ MarHV after Ic inoculation (LD50 of 62,000 and 32,000 p.f.u., respectively, for viruses 5D-6B and 5D-4B) and gave no fatalities after Sc inoculation. Mice surviving TK- MarHV infections were protected from challenge with TK+ parental MarHV. Recombinant TK- MarHV's may be useful as vectors for the expression of foreign genes in animal cells and as the starting material for the design of vaccines.