The increasing prevalence of atopic diseases over the last few decades is thought to be due to reduced exposure to environmental microbes that normally down-regulate allergic responses (hygiene hypothesis). We have shown previously that administration of the environmental microbe Mycobacterium vaccae ameliorates atopic dermatitis in school-age children at 3 months post-treatment. The present study tested the hypothesis that M. vaccae suppresses Th2-type cytokine activity and increases transforming growth factor (TGF)- β1 immunomodulatory activity in these children. Interleukin (IL)-4, IL-5, TGF-β1 and interferon (IFN)-γ activity were assessed in resting and stimulated peripheral blood mononuclear cells (PBMC) isolated from 12 of the children who received M. vaccae in our original clinical trial. A cDNA expression array was used to examine a broader range of cytokine pathway transcripts. There were no significant changes in either Th2-type or TGF-β1 activity. A 5- to 10-fold increase in Th1-type activity was found at 1 month post-M. vaccae administration (P <0.05), but it had returned to baseline by 3 months. The results do not support the hypothesis that M. vaccae reduces Th2-type or increases TGF-β1 activity of PBMC isolated from children with atopic dermatitis. The transient surge in IFN-γ at 1 month is unlikely to explain any improvement in eczema score at 3 months. © 2005 British Society for Immunology.