The beta-chemokine RANTES, a T-lymphocyte activator, chemoattractant, and inducer of homotypic aggregation, is considered to exert extensive effects on T lymphocytes through either G protein-coupled or protein tyrosine kinase (PTK) signaling pathway. In the present study, we analyzed RANTES-induced signal transduction through PTK as an early event in T-lymphocyte activation. Tyrosine phosphorylation is detected by immunoblots in the human T-cell line H9 after incubation with human recombinant RANTES. The tyrosine phosphorylation of a protein with a molecular mass of about 25 kD is measurable as early as 30 s and maximal at 1-5 min; and is a dose-dependent effect. The phosphorylation response can be abrogated by the tyrosine-kinase inhibitor herbimycin A (HA) but is insensitive to heterotrimeric Galphai protein inhibitor pertussis toxin (Ptx). This phenomenon is also observed in a visible homotypic aggregation response after incubation serum-starved H9 cells with RANTES. The phosphorylation response can not be down-regulated by preincubation with either anti-CC chemokine receptor 5 (CCR5) antibody or HIV-1Bal supernatants. Our results suggest that tyrosine phosphorylation of a protein with molecular mass of about 25 kD via Src-family PTK(s) is an early event in T-lymphocyte activation associated with the homotypic aggregation in response to RANTES.