Herpes simplex virus (HSV) establishes a lifelong infection in humans. Reactivation of latent virus occurs intermittently so that the immune system is frequently exposed to viral Ag, providing an opportunity to evaluate memory T cells to a persistent human pathogen. We studied the persistence of genital herpes lesion-derived HSV-specific CD8+ CTL from three immunocompetent individuals with frequently recurring genital HSV-2 infection. All CTL clones were HSV-2 type specific and only one to three unique clonotypes were identified from any single biopsy specimen. The TCRBV genes utilized by these clonotypes were sequenced, and clonotype-specific probes were used to longitudinally track these clonotypes in PBMC and genital lesions. CTL clonotypes were consistently detected in PBMC and lesions for at least 2 and up to 7 years, and identical clonotypes infiltrated herpes lesions spaced as long as 7.5 years apart. Moreover, these clones were functionally lytic in vivo over these time periods. Additionally, CTL clones killed target cells infected with autologous viral isolates obtained 6.5 years after CTL clones were established, suggesting that selective pressure by these CTL did not result in the mutation of CTL epitopes. Thus, HSV recurs in the face of persistent CD8+ CTL with no evidence of clonal exhaustion or mutation of CTL epitopes as mechanisms of viral persistence.