Forty-seven Mycobacterium tuberculosis genes from the 'regions of difference' RD2-7, RD9-13 and RD15 were cloned and expressed, and the purified recombinant proteins were screened for their serodiagnostic potential. Evaluation of six selected proteins in serum samples from Danish resident tuberculosis patients and healthy controls led to identification of Rv0222 as the most promising serodiagnostic antigen. Recognition of the Rv0222 was compared with the 38 kDa protein and a fusion protein of the RD1 proteins ESAT-6 and CFP10 in a serum panel from pulmonary tuberculosis (TB) patients from Uganda. The highest overall sensitivity was observed for Rv0222 compared to BCG-vaccinated non-endemic healthy controls as well as symptomatic endemic controls. Importantly, Rv0222 identified human immuno deficiency (HIV) virus-positive patients and HIV-negative patients with the same overall sensitivity. The results emphasize the importance of cut-off values in TB endemic regions based on endemic control individuals to diagnose active TB, and identify Rv0222 as a promising new antigen for serodiagnosis of TB in both HIV-negative and HIV-positive patients.