Previously, we have shown that both T and B lymphocytes from chronically nicotine-treated (NT) animals exhibit tolerance to activation by Ags (ligation of Ag receptors), as indicated by their decreased ability to mobilize intracellular calcium and, at least in T cells, arrest of cells in the G0/G1 phase of the cell cycle. Herein, we demonstrate that NT T cells significantly lose their ability to up-regulate inositol trisphosphate synthesis in response to TCR ligation or nonspecific activation of G proteins by AIF-4. However, increases in cAMP concentrations of T cells following activation of G protein-sensitive adenylate cyclase by cholera or pertussis toxin were not significantly affected by the nicotine treatment. Interestingly, compared with control T cells, the background levels of inositol trisphosphate were significantly elevated in NT T cells, indicating some degree of activation in these cells. This inference was further supported by observations that naive T cells from NT animals exhibit tyrosine phosphorylation of several substrates, including phospholipase C-gamma1, which were either absent or underphosphorylated in unstimulated control T cells. Moreover, when, after 4-wk nicotine treatment, nicotine pumps were removed and serum cotinine levels fell to background, inhibition of the Ab-forming cells and Ca2+ responses continued for at least 2 more wk. These results suggest that chronic in vivo nicotine exposure leads to T cell anergy and may contribute to nicotine/cigarette smoke-induced immunosuppression.