Cytokine production by human lymphocytes from healthy children (ages 0-18 years) was assessed using a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. To establish valid cytokine values for intracellular cytokine expression in healthy children in the different age groups, we measured 117 samples after 24 h in vitro stimulation with PMA, ionomycin and brefeldin followed by staining with intracellular anti-cytokine and surface antibodies. We found decreasing IL-2 expression, increasing IFN-gamma and TNF-alpha production and stable IL-4, Ki67 and TGFb levels with advancing age. The cytokines were mainly produced by memory T-cells. Apart from age, there was a differential expression in boys and girls: boys (< 6 years) produce significantly more IL-2 (p < 0,04), while girls > 12 years produce more IFNg than boys of the same age (p < 0.05). This systematic analysis of cytokine profiles during childhood allows a better understanding of immune maturation and will contribute significantly to the interpretation of cytokine data from children with pathological conditions.