Three herpes simplex virus type I (HSV-I) glycoproteins of apparent molecular masses 103, 63, and 115 kD have been purified using virus-specific monoclonal antibodies (mAb) G8D1, C2D2, and T157, respectively. Both G8D1 and C2D2 neutralize HSV-I in vitro and passively protect CBA mice against HSV-I infection in vivo, whereas T157 is neither neutralizing nor passively protective. However, mice given a single subcutaneous injection of 30 micrograms 115 kD glycoprotein in saline were completely protected against lethal challenges of HSV-I administered intraperitoneally or in the footpad 7 d after immunization. In contrast, mice similarly immunized with 103 or 63 kD glycoproteins were only partially protected. The prophylactic immunity was correlated with an early induction of specific antibody, which became even more evident 3 d after virus challenge. There was a remarkable similarity in antibody isotype distribution between the responses to 115 kD glycoprotein and to heat-inactivated intact HSV-I. However, the prechallenge sera from 115 kD glycoprotein hyperimmunized mice were again neither virus-neutralizing nor passively protective. All three glycoproteins induced only low levels of delayed-type hypersensitivity (DTH). Pretreatment of mice with cyclophosphamide significantly enhanced DTH to 115 kD and 103 kD glycoproteins in the absence of antibody, but failed to confer significant immunity, indicating that DTH alone is insufficient for protection. Splenic and lymph node Ig- (B cell-depleted) cells from mice protectively immunized with 115 kD glycoprotein could adoptively transfer effective protection and enhance a virus neutralizing antibody response in normal recipients challenged with a lethal dose of HSV-I. Both the protection and the ability to enhance neutralizing antibody were diminished when the cells were treated with mAb GK 1.5 and complement. These results therefore demonstrate that the 115 kD glycoprotein, though not apparently containing accessible epitopes for the induction of virus-neutralizing antibody, possesses determinants capable of activating helper T cells. These L3T4+ cells confer strong protective immunity by enhancing protective antibody upon challenge infection, probably through associative help.