Localization of memory CD8+ T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX 3 CR1 distinguishes memory CD8+ T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3 CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8+ T cells with effector function. We find CD62L hi CX3 CR1+ memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1+ memory CD8+ T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3 CR1-based functional classification will help to resolve the principles of protective CD8+ T-cell memory. 2015 Macmillan Publishers Limited. All rights reserved.