Heparin-binding haemagglutinin (HBHA) is an important Mycobacterium tuberculosis virulence factor. It displays a complex methylation pattern in its C-terminal, functional domain, which protects this domain against proteolysis. Here, it is shown that HBHA methylation is catalysed by mycobacterial enzymes and a radio-enzymatic and a nonradioactive enzyme assay are described, based on the recognition of methylated HBHA by monoclonal antibodies. MS analysis of in vitro methylated HBHA shows a complex methylation pattern similar to that of naturally methylated HBHA. Using recombinant hybrid molecules as acceptor substrates, it was found that the N-terminal domain of HBHA is not required for recognition by the HBHA-methyltransferase(s), although it is required for in vivo methylation.