To investigate the potential role of neutrophils in initiation of immune responses to mycobacteria, we have characterized the response of human neutrophils to infection with Mycobacterium bovis bacille Calmette Guerin, the BCG vaccine. BCG induced transcription and secretion of the chemokine CXCL8, by signalling through Toll-like receptors TLR2 and TLR4, in conjunction with the adaptor protein myeloid differentiation factor 88 (MyD88). Blocking of responses with antibodies revealed a difference in the kinetics of signalling through the different TLRs. Anti-TLR2 antibody blocked the early phase of CXCL8 and MyD88 induction. Anti-TLR4 antibody blocked the late phase of induction occurring 2 h after infection. The existence of a TLR/MyD88 pathway for recognition and response to mycobacterial ligands provides neutrophils with the ability to drive the recruitment and activation of inflammatory cells during the early phase of mycobacterial infection and immunization.