Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne's disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutininP (PHAP), pokeweed mitogen (PWM), and E. coli lipopolysaccharide (LPS). In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria to cell ratio. After incubation, cell-free supernatants were analyzed for g-interferon (g-IFN) activity. Cells from subclinical cows produced significantly with mitogens, ConA, PHAP, and PWM. Levels of g-IFN were produced by cells isolated from subclinical animals compared to cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that g-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinical animals.