Two antigens from Mycobacterium avium subsp. paratuberculosis (M. a.paratuberculosis) and Mycobacterium avium subsp. silvaticum (M. a.silvaticum) were employed in two different assays to measure the cell-mediated immune reactivity of ovine peripheral blood lymphocytes. Using a standard lymphocyte stimulation assay, proliferative responses to purified protein derivative (PPD) of M. a.paratuberculosis were detected but did not discriminate a) between immunized and infected animals or b) between animals infected with M. a.paratuberculosis and those infected with M. a.silvaticum. In contrast, with defined antigens, responses to a purified 30 kDA alpha antigen from M. a.paratuberculosis were observed only with lymphocytes from sheep inoculated with live organisms whereas a P40 protein from M. a.silvaticum elicited in vitro responses only with lymphocytes derived from sheep inoculated with live M. a.silvaticum. When a sandwich enzyme-linked immunosorbent assay was used to detect the release of interferon-gamma, responses were observed when lymphocytes from animals given live mycobacteria were cultured in the presence of a 30 kDa antigen. With lymphocytes from immunized animals responses were only slight. P40-induced release of interferon-gamma was observed only with lymphocytes from animals given live M. a.silvaticum. Lymphocytes from 2 sheep serving as negative controls did not respond to any of the antigens in either of the assays.