Limited specificity of the tuberculin skin test incited the development of in vitro assays based on Mycobacterium tuberculosis-specific antigens such as ESAT-6 that are lacking in Bacillus Calmette Guérin (BCG). In animal studies, intradermal ESAT-6 was safe and induced specific skin test responses. The aim of the study was to assess the safety of intradermal recombinant dimer ESAT-6 (rdESAT-6) compared with tuberculin and to determine the human dose. The study design was a double-blind Phase I study with intra-subject randomization to the left and right forearm, comparing 2 Tuberculin Units (TU) intradermal tuberculin (RT23) with 0.01, 0.1, 1 or 10μg rdESAT-6 in groups of five healthy controls or treated tuberculosis (TB) patients. The risk of sensitization after skin testing was assessed in healthy volunteers. All doses were tolerated well by healthy volunteers and responses to rdESAT-6 were limited to transient redness after 24h only at the highest dose. No sensitization was observed. Because 1μg rdESAT-6 induced large responses with local side effects in some TB patients, the 10μg dose of rdESAT-6 was not tested. Mean responses to 0.01, 0.1 and 1μg rdESAT-6 measured 14.0, 19.8 and 38.8mm of redness, respectively, and 7.0, 13.4 and 14.6mm of induration. The response to tuberculin was similar to the responses to 0.1μg rdESAT-6. Mild local side effects due to tuberculin and rdESAT-6 were observed in 8/15, respectively, 6/15 patients, more pronounced at the highest rdESAT-6 dose. In conclusion, this pilot Phase I study of safety, feasibility and dose finding of intradermal rdESAT-6 provides proof of principle of a specific skin test for human use. No serious adverse events were observed but the study was not sufficiently powered to demonstrate complete safety. Intradermal rdESAT-6 did not seem to sensitize healthy volunteers. In treated TB patients, responses to rdESAT-6 were optimal at 0.1μg. Further studies are needed to evaluate sensitization after repeated doses and to study the effect of additional CFP-10 on the sensitivity of a TB-specific skin test.