The development of novel vaccine strategies to replace or supplement bacille Calmette-Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine-induced levels of cellular immunity were assessed by determining interferon-γ (IFN-γ) responses in vitro. Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-γ-secreting cells than the two viral vectors used in combination (P = 0.0055), or BCG used alone (groups 2 and 3, P = 0.04). The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG/MVA85A heterologous prime-boost vaccination were compared with BCG/BCG homologous revaccination. The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG/BCG-vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.