Granulysin (GNLY) is a cytolytic/apoptotic molecule highly expressed in immune cells, particularly NK cells, at the maternal-fetal interface. The primary function of GNLY is to carry out lysis or apoptosis induction in target cells, tumor cells or cells infected by intracellular pathogens. To exert some of its functions GNLY needs to collaborate with perforin. The purpose of this study was to determine: (a) the expression of GNLY at the gene and protein levels at the maternal-fetal interface, (b) the relationship(s) between GNLY and perforin, and (c) GNLY secretion by NK cells stimulated by the NK-sensitive K562 cell line and its HLA-C and HLA-G transfectants. GNLY and perforin genes were found to be highly activated at the interface. GNLY mRNA was present at significantly higher levels compared with other cytolytic/apoptotic molecules. Confocal microscopy analysis showed that most first trimester pregnancy decidual lymphocytes simultaneously contained both GNLY and perforin protein in their cytoplasm, with a punctuate pattern consistent with granule localization. In contrast to peripheral blood, in unstimulated decidual lymphocytes GNLY and perforin rarely co-localized (10% of GNLY-positive cells and 20% of perforin-positive cells were positive for both proteins). Contact between decidual lymphocytes and K562 cells caused GNLY and perforin to be expressed in the same granules (approximately 50% co-localization), i.e., to attain the pattern seen in peripheral blood lymphocytes. The abundant GNLY secretion by decidual NK cells compared with peripheral blood NK cells after 2h of contact with the NK-sensitive K562 cells and K562 transfectants was striking.