Evidence is emerging that moonlighting proteins, defined as proteins with more than one biological function, play important roles in bacterial virulence. The Mycobacterium tuberculosis chaperone, chaperonin 60.1, is a potent stimulator of human monocyte cytokine synthesis and modulator of giant cell and osteoclast formation. Previously, we had shown that these moonlighting activities resided in the equatorial domain of this protein. In this study, through the generation of chaperonin 60.1 amino acid sequence-deletion mutants and synthetic peptides, we have identified the minimal moonlighting site in this molecular chaperone responsible for monocyte activation as peptide sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, residues 461–491, in the equatorial domain, Modelling of this biologically active sequence in the M. tuberculosis chaperonin 60.1 protein reveals a surface-exposed motif with significant α-helical structure.