We report the construction of a Marek's disease virus (MDV) mutant containing the lacZ gene of Escherichia coli inserted into a homologue of the US6 (glycoprotein D, gD) gene of herpes simplex virus. The mutant was constructed using the high-passage GAatt85 MDV strain as the parent virus, since that strain grows readily in chicken embryo fibroblasts using culture conditions conducive to mutant virus construction. The lacZ insertion site was positioned one third of the way into the US6 (gD) open reading frame. Insertion of the lacZ gene disrupted a major 6.2 kb transcript that initiated approximately 2.5 kb upstream of the gD homologue gene in the vicinity of the US3 homologue and sorf4 genes, and extended into the US7 (gI) homologue gene. The mutant virus (US6lac) and the parent virus had similar growth kinetics in cell culture at 37 degrees C and 41 degrees C. Furthermore, the US6lac mutant could be reisolated from the spleens and peripheral blood of infected chickens with a frequency comparable to that of the parent virus. Our results indicate that the gene encoding the gD homologue is nonessential for growth in cell culture or for infection of chickens following intra-abdominal inoculation with an attenuated serotype-1 MDV.