Optimal culture conditions were established for the analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression and protein production, as well as proliferative capacity of peripheral blood mononuclear cells (PBMC). These culture conditions permitted the analysis of differences in the responses of house-dust mite (HDM) allergic patients and healthy controls after polyclonal and allergen-specific stimulation. Proliferative responses were optimal when PBMC were cultured in RPMI, whereas for studying mRNA expression by RT-PCR and protein production by ELISA, PBMC should be stimulated in Yssels's medium. Blood holding period influenced the cytokine mRNA expression and proliferative capacity of primarily the unstimulated cells. It is thus crucial to isolate PBMC as soon as possible, and in any event no later than 7 hours after blood collection. Proliferative responses to Dermatophagoides pteronyssinus-extract were observed in HDM allergic patients (mean stimulation index (SI) = 5.3+/-0.75), but not in non-allergic subjects (mean SI = 2.3+/-0.21). After D. pteronyssinus-specific stimulation, IL-4 mRNA expression was significantly (p = 0.03) increased in HDM-allergic subjects compared to non-allergic subjects. No significant differences were found in IFN-gamma mRNA expression between HDM-allergic and non-allergic subjects. Both IFN-gamma (p = 0.04) and IL-4 (p = 0.06) protein production were increased after D. pteronyssinus-specific stimulation in HDM-allergic subjects compared to non-allergic subjects. Our data suggest activation of both Th1 and Th2-like cells, as well as CD8+ T cells in allergic patients. Furthermore, analysis of possible functional differences in PBMC between allergic and non-allergic patients, necessitates polyclonal and allergen-specific stimulation of PBMC. Moreover, proliferative responses as well as cytokine mRNA expression and protein production should be studied under optimal culture conditions to highlight the often subtle differences.