CD8+ cytolytic T lymphocytes (CTL) are likely to be an important component of effective vaccines against human immunodeficiency virus type 1 (HIV-1). CTL can be induced most effectively with live virus vectors. However, because of concerns about the safety of such vectors, a nonreplicating canarypox vector (ALVAC) capable of expressing foreign genes in mammalian cells has been developed. This study evaluated the capacity of an ALVAC vector expressing the HIV-1MN envelope (env) glycoprotein to induce HIV-1-specific CTL in seronegative volunteers. Protocols were designed to determine whether immunization with ALVAC alone or in combination with subunit boosting could induce CTL in vaccinia-immune and -naive volunteers. A simple method for antigen-specific in vitro stimulation was used to detect CTL responses in HIV-1-seronegative vaccine recipients. The results indicate that low doses of a nonreplicating virus vector alone can elicit both CD4+ and CD8+ HIV-1-specific CTL in a subset of seronegative volunteers.