A construct containing the gene for glycoprotein D of herpes simplex virus (HSV-gD), under the control of the simian virus 40 early promoter, was microinjected into single-cell embryos, and four transgenic mouse lines were established. Three were homozygous (lines 75, 111, and 113) and one was hemizygous (line 108) for the HSV-gD gene. Examination of sera revealed that only one of the lines (line 75) spontaneously produced antibody to HSV-gD. Immunization of the other three lines with vaccinia virus-HSV-gD showed that one of them (line 113) responded by making antibody to HSV-gD, whereas the other two (lines 108 and 111) appeared to be immunologically tolerant. Evidence that tolerance was not absolute was obtained by immunization with infectious HSV, which resulted in an antibody response to HSV-gD in some of the animals from line 111. Examination of organs for HSV-gD mRNA revealed transcripts in the tolerant line (line 108) and in the partially tolerant line (line 111), but not in the nontolerant line (line 113), suggesting that the development of immunological tolerance requires active expression of the HSV-gD gene.