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 antigenin-dependent activator of T cells in vitro.