Histamine shifts TH1/TH2 cytokine balance from TH1 to TH2 cytokines and regulates the function of lymphocytes after binding to histamine receptors. The phosphorylation of STAT factors and the translocation to the nucleus are important steps in the regulation of TH1/TH2 cytokine balance. This study was designed to investigate the effects of histamine on the phosphorylation of STAT4. C57BL/6 splenocytes were isolated and treated with histamine (10⁻⁴ to 10⁻⁹ M) after activation with either PMA (phorbol 12 myristate 13-acetate) plus ionomycin or IL-12. The phosphorylated STAT4 levels were analyzed by Western Blot Analysis. Unstimulated splenocytes expressed both STAT4 and phosphorylated STAT4. However, phosphorylated STAT4 gradually declined within 24 h. Histamine increased the phosphorylation of STAT4 at lower concentrations (10⁻⁶ to 10⁻⁹ M), and had no effect at higher concentrations (10⁻⁴ and 10⁻⁵ M) after the cells were stimulated with PMA+ionomycin. Histamine did not affect IL-12-induced phosphorylation of STAT4. To characterize the histamine receptor subtypes involved in the up-regulation of STAT4 phosphorylation, various H₁, H₂ and H₃/H₄ receptor antagonists and/or agonists were employed. H₁ receptor agonist (betahistine), but not H₂ receptor agonist (amthamine), induced phosphorylation of STAT4. H₁ receptor antagonist (pyrilamine) inhibited histamine-mediated phosphorylation of STAT4. However, H₂ receptor antagonist (ranitidine) and H₃/H₄ receptor antagonist (thioperamide) did not alter this effect. Tyrosine kinase inhibitor (tyrphostin) failed to block histamine-mediated phosphorylation of STAT4. These observations suggest that histamine up-regulated the phosphorylation of STAT4 via H₁ receptors, and that the Ca²⁺-PKC pathway, but not the tyrosine kinase pathway, was involved in this effect.