Macrophages are phagocytes that recognize bacteria and subsequently activate appropriate innate and adaptive immune responses. TLRs are essential in identifying conserved bacterial structures and in initiating and mediating innate immune responses. In this work, we have characterized TLR gene expression in human monocyte-derived macrophages in response to stimulation with two live Gram-positive bacteria, a human commensal and probiotic Lactobacillus rhamnosus GG (LGG), and an important human pathogen Streptococcus pyogenes. LGG and S. pyogenes enhanced TLR2 expression in macrophages. LGG and S. pyogenes also required TLR2 for NF-kappaB activation. Only pathogenic S. pyogenes was able to up-regulate TLR3 and TLR7 gene expression. This up-regulation was dependent on IFN-alpha/beta, as neutralizing anti-IFN-alpha/beta antibodies reduced S. pyogenes-induced TLR3 and TLR7 mRNA expression. Our results show that despite similarities, TLR responses of macrophages differ for a Gram-positive probiotic and a pathogen. Our data suggest that macrophages can discriminate between probiotic and pathogenic bacteria by IFN-mediated TLR gene regulation.