Previous studies have demonstrated that STAT5 is critical for expression of granulysin and antimicrobial activity. Because the signaling pathway and the resultant microbicidal activity are defective in HIV-infected patients, the mechanism by which STAT5 leads to granulysin expression is of great interest. In the current study, IL-2-stimulated CRL-2105 CD4(+) T cells expressed granulysin and killed Cryptococcus neoformans similar to primary CD4(+) T cells. The enhancer activity of the upstream element of the granulysin promoter was analyzed in primary CD4(+) T cells and CRL-2105 T cells with a luciferase reporter assay, and a STAT5 binding site, 18,302 to 18,177 bp upstream of the transcription start site, was identified as an enhancer. Additionally, the enhancer functioned in the context of heterologous SV40 promoter irrespective of its transcriptional orientation. Chromatin immunoprecipitation and EMSAs demonstrated that the enhancer element bound STAT5 both in vivo and in vitro, and mutation of the STAT5 binding site abrogated its enhancer activity. Furthermore, overexpression of a dominant negative STAT5a abolished the enhancer activity of the STAT5 binding site and abrogated the anticryptococcal activity of IL-2-stimulated primary CD4(+) T cells. Taken together, these data provide details about the complex regulation leading to granulysin expression and anticryptococcal activity in primary CD4(+) T cells.