The purpose of this work was to investigate the interaction of the pro-inflammatory cytokine, IFN-gamma, and the anti-inflammatory cytokines, TGF-beta and IL-10, in the subclinical and clinical stages of Johne's disease. A retrospective quantitative analysis of IFN-gamma, IL-10 and TGF-beta mRNA gene expression in tissues such as ileum, ileal-cecal junction, ileal-cecal node, and mesenteric lymph node obtained from cows naturally infected with Mycobacterium paratuberculosis and healthy non-infected control cows revealed an upregulation of IL-10 and TGF-beta in tissues obtained from clinically infected animals but not in tissues obtained from healthy or subclinically infected cows. Analysis of IFN-gamma mRNA gene expression in these tissues showed that expression of this cytokine is upregulated only in subclinical cows. We further evaluated the effects of exogenous IFN-gamma, IL-10 and TGF-beta in an extended culture system (7--12 days) using unfractionated PBMC cultures from naturally infected and healthy non-infected cows after infection with live M. paratuberculosis. We determined that the presence of IL-10 and TGF-beta in culture supernatants was correlated with decreased ability of monocyte-derived macrophages in the unfractionated cell cultures to kill M. paratuberculosis in vitro, with a resultant decrease in IFN-gamma production in these cultures. IL-10 and TGF-beta were upregulated in response to in vitro infection in cell cultures from infected animals regardless of their disease status. Prior to in vitro infection with M. paratuberculosis TGF-beta levels were lower in subclinically infected animals compared to clinically infected or healthy animals. After in vitro infection, TGF-beta levels in culture supernatants of subclinically infected animals were similar to that of healthy animals. This is the first study to investigate the interactive roles of IFN-gamma, IL-10 and TGF-beta in natural and experimental M. paratuberculosis infections. The data presented here suggest an important regulatory role of anti-inflammatory cytokines IL-10 and TGF-beta on IFN-gamma production and the ability of macrophages to kill M. paratuberculosis in vitro.