Leishmania species present unusual challenges to the immune system with their capacity to downregulate inflammatory responses as well as their ability to live within macrophages. Although toll-like receptor (TLR) pathways have been implicated in the recognition of several classes of pro-inflammatory microbes, it is not known if pathogens with anti-inflammatory properties activate the host response through this family of proteins. In this study, Leishmania major stimulation of cytokine promoter-luciferase reporter constructs was examined in transfected macrophages to detect early signs of cellular activation. L. major selectively activated the promoter region of IL-1 alpha, but not IL-6, IL-8, IL-10, or an NF-kappa B reporter. IL-1 alpha mRNA expression was also stimulated by L. major, although at lower levels than lipopolysacharide-stimulated macrophages. No IL-1 alpha protein was detectable in stimulated cell lysates or culture supernatants. Transfection of macrophages with a dominant-negative version of myeloid differentiation factor 88 (MyD88), an adaptor protein which interacts with TLRs, inhibited activation of the IL-1 alpha promoter. Furthermore, stimulation of IL-1 alpha RNA expression by L. major was inhibited in peritoneal macrophages from MyD88-/- as compared to MyD88+/+ mice. These observations indicate that L. major stimulates IL-1 alpha promoter activity and mRNA expression in macrophages through MyD88-dependent pathways. However, additional anti-inflammatory pathways must also be activated which downregulate transcription and ultimately inhibit translation of the IL-1 alpha protein. Examination of promoter activation is a powerful tool for understanding the early events in macrophage activation for anti-inflammatory pathogens such as Leishmania that have mechanisms to downregulate transcription and translation.