Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10. 4. In most infected humans, TB10. 4 is strongly recognized, raising interest in TB10. 4 as a potential vaccine candidate and substitute for ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10. 4 induced a significant protection against tuberculosis. Fusing Ag85B to TB10. 4 produced an even more effective vaccine, which induced protection against tuberculosis comparable to bacillus Calmette-Guérin vaccination and superior to the individual Ag components. Thus, Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for TB10. 4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials.