In human lungs, the earliest encounter of Mycobacterium tuberculosis, the agent of tuberculosis, involves alveolar epithelial cells. Droplets expectorated by a patient with tuberculosis are likely to contain a mixed population of M. tuberculosis cells in different physiologic and metabolic states from the lung lesions of the patient. Here, we compared the chemokine expression patterns of human epithelial cell line A549 and RAW 264.7 macrophage cells infected with wild-type M. tuberculosis H37Rv against patterns induced by a mutant that accumulates free mycolic acids in its cell wall (Dmce1). We also examined the effect of free mycolic acids on toll-like receptor-2 (TLR-2). Wild-type M. tuberculosis induced significantly higher levels of IL-8, MCP-1, RANTES, and IP-10 in both cell types than did Dmce. Free mycolic acids reduced the ability of the mammalian cells to respond to a TLR-2 agonist in a dose-dependent manner. These observations suggest that differences in mycolic acid abundance in the M. tuberculosis cell wall can affect TLR-2-mediated pro-inflammatory response in both epithelial and macrophage cells. The final fate of a new infection may be ultimately determined by the proportion of M. tuberculosis cells expressing free mycolates in the infecting inoculum population.