Upon stimulation, leukocytes secrete chemokines to attract distinct effector cell populations to the site of inflammation. Only a few data are available about the phenotype and the frequencies of cells expressing particular chemokines. To date, the expression of individual chemokines is mainly analyzed at the mRNA level or via ELISA. Both techniques do not allow the analysis of chemokines at the level of single cells. We have established the intracellular flow-cytometric detection of the murine chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES) and activation-induced, T cell-derived and chemokine-related cytokine (ATAC)/lymphotactin. For detection of the nonclassical chemokine ATAC, we generated the novel mAb MTAC-2. Using this assay, we analyzed for the first time the frequency and kinetics of the expression of these murine chemokines in lymphocyte subpopulations. We show that these chemokines are differentially expressed by NK cells, naive and memory CD4(+) and CD8(+) T cells. Our results emphasize that the analysis of chemokine expression at the single-cell level is required to understand the functional role of specialized lymphocyte subpopulations in vivo.