A major objective of current HIV-1 vaccination strategies is the induction of HIV-1-specific CD8+ MHC class I-restricted CTL responses, which are suggested to play a pivotal role in viral clearance and protection against HIV-1 disease progression. However, the marked genetic diversity of HIV-1 and existence of distinct viral subtypes or clades could potentially hinder the development of a universally efficacious HIV-1 vaccine. In this study we examined HIV-1 intraclade (B(LAI) versus B(MN)) Env gp160-specific CTL reactivity in recently HIV-1 clade B-infected individuals. We further evaluated the extent of interclade CTL cross-recognition of the divergent A and C Env gp160 subtypes, that are highly prevalent in the global pandemic. Freshly isolated PBMCs were stimulated in vitro with autologous PBMCs infected with recombinant vaccinia vectors expressing HIV-1 env, gag, pol, and nef genes derived from HIV-1 clade B. All 13 of the 19 HIV-1-seropositive subjects who elicited significant clade B Env gp160LAI CD8+ CTL responses also demonstrated comparable levels of CTL cross-reactivity against clade C92BR025 Env gp160. Nine of these individuals also showed extensive interclade CTL cross-recognition of clade A92UG037 Env gp160. Two HLA class I B7 donors had nondetectable intraclade CTL response against B Env gp160MN, while generating significant intraclade B(LAI) and interclade (A and C) Env gp160 CTL cross-reactivity. These observations serve to underscore the central importance of the HLA background of individuals in determining the pattern of immune reactivity to natural HIV-1 infection and presumably vaccines. Five donors studied also demonstrated broad CTL cross-reactivity against clade A92UG037 Gag p55, Pol, and/or Nef antigens. In conclusion, this present study indicates that there is a considerable degree of CD8+ CTL cross-recognition of the highly divergent HIV-1 Env gp160 subtypes during early phases of HIV-1 infection. Such findings suggest that HIV-1 vaccines based on a single clade that can induce extensive cross-clade immunity may demonstrate utility in diverse geographical regions.