The chemokine RANTES induced biphasic mobilization of Ca²⁺ in T cells. The initial peak, a transient increase in cytosolic Ca²⁺ mediated by a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled pathway, was associated predominantly with chemotaxis. The second peak, Ca²⁺ release and sustained influx dependent on protein tyrosine kinases, was associated with a spectrum of cellular responses—Ca²⁺ channel opening, interleukin-2 receptor expression, cytokine release, and T cell proliferation—characteristic of T cell receptor activation. Other chemokines did not produce these responses. Thus, in addition to inducing chemotaxis, RANTES can act as an antigen-independent activator of T cells in vitro.