The objective of the present study was to assess whether bovine herpesvirus 4 (BHV-4) is able to infect in vitro-produced bovine embryos. A green recombinant BHV-4 (BHV-4EGFP deltaTK), obtained by insertion of an EGFP gene into the TK locus of BHV-4, was used. The presence of this marker protein made it possible easily to detect infected cells under physiological conditions, without harmful manipulation of the cells or the addition of exogenous substrates, so that the spread of the virus could be followed in real time. Zona pellucida intact (ZP-I) and zona pellucida open (ZP-O) blastocytes were exposed to 10(6) TCID50 viral particles and infection was monitored by fluorescent microscopy for 48 h. Expression of EGFP and degeneration of embryonic cells was observed in three of the 18 ZP-O embryos, but in none of the ZP-I embryos. It was concluded from this preliminary study that BHV-4 has only a low ability to infect in vitro-produced bovine embryos, depending on the absence of ZP, the amount of virus present and the stage of embryonic development. However, embryonic stem cells could be transduced by BHV-4EGFP deltaTK just after differentiation, as shown by expression of EGFP.