Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are critical in the development of an effective immune response. Vitamin D, essential in short-term calcium homeostasis and recently shown to modulate proliferation and function of blood mononuclear cells from adult dairy cattle, may be an effective modulator of the calf's immune system. Effects of antigen sensitization and 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] on cytokine secretion by cells from calves vaccinated with Bacille Calmette-Guérin (BCG) were examined. One-week-old dairy calves (n = 6) and yearling heifers (n = 4) were vaccinated concurrently with BCG and boosted six weeks later. Ten weeks after primary vaccination, cells from vaccinated calves and adults, and nonvaccinated, age-matched calves (n = 4) were evaluated in vitro for their capacity to produce IFN-gamma and TNF-alpha. Cells were stimulated with pokeweed mitogen (PWM) or recall antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] in the presence of 0, 0.1, 1.0, and 10 nM of 1,25-(OH)2D3 for 20, 44, and 68 hours, respectively. IFN-gamma and TNF-alpha concentrations in culture supernatants harvested at these times were quantified by enzyme-linked immunosorbent assay (ELISA). PPD-induced IFN-gamma and TNF-alpha responses of cells from vaccinated calves and adults were greater than responses of autologous unstimulated cells. In contrast, PPD-specific responses of calf and adult cells collected immediately before primary vaccination were substantially lower and comparable to responses in resting (i.e., unstimulated) cultures. At ten weeks, the PPD-specific response of vaccinates exceeded the response of nonvaccinated calves; however, responses of vaccinated calves were more vigorous than corresponding responses of vaccinated adults. Incubation period also influenced the magnitude of both IFN-gamma and TNF-alpha, responses in PPD- and PWM-stimulated cultures. Effects of 1,25-(OH)2D3 on antigen-induced secretion of IFN-gamma and TNF-alpha were marginal. Only IFN-gamma responses of vaccinated adults were affected by 1,25-(OH)2D3. Vitamin D caused a concentration-dependent decrease in IFN-gamma response and an increase in TNF-alpha response in PWM-stimulated cultures. These results indicate that animal maturity (i.e., age) and antigenic experience affect IFN-gamma and TNF-alpha secretion by bovine leukocytes and suggest that 1,25-(OH)2D3 can alter secretion of both cytokines under specific conditions of culture.