T cells were exposed to various concentrations of nicotine or smokeless tobacco extract (STE) during in vitro immune responses in order to examine effects upon expression of T cell costimulatory counter-receptors, CD28 and CTLA-4, and cytokine production. Splenic mononuclear cells (SPM) were exposed to 1:10(2) to 1:10(3) dilutions of STE or 1-100 micrograms/ml nicotine during 48 and 72 h of stimulation with anti-CD3. Expression of CD28 and CTLA-4 was evaluated with flow cytometry and production and expression of IL-2, IL-4, IL-10 and IFN-gamma were evaluated using ELISA and RT-PCR. The data here indicate that the percentage of CD4+ T cells expressing CD28 declined while percentage and intensity of CTLA-4 expression increased with exposure to a 1:10(2) dilution of STE during the T cell response. Exposure to nicotine decreased the percentage of CD4+ T cells expressing both CD28 and CTLA-4 and decreased the intensity of CD28 expression. Responding T cells exposed to nicotine produced significantly less Th1 cytokines, IL-2 and IFN-gamma, but significantly more Th2 cytokines, IL-4 and IL-10. Cytokine specific mRNA expression was only slightly affected by the exposure to nicotine. Thus, exposure of T cells to physiological concentrations of STE or nicotine can alter the T cell expression of CD28 and CTLA-4, and the CD4 T cell cytokine expression pattern.