Herein we report the synthesis and activity of an enzyme-directed immunostimulant with immune cell activation mediated by -galactosidase, either exogenously added, or on B16 melanoma cells. Covalent attachment of a -galactopyranoside to an imidazoquinoline immunostimulant at a position critical for activity resulted in a pro-immunostimulant that could be selectively converted by -galactosidase into an active immunostimulant. The pro-immunostimulant exhibited -galactosidase-directed immune cell activation as measured by NF-B transcription in RAW-Blue macrophages or cytokine production (TNF, IL-6, IL-12) in JAWSII monocytes. Conversion of the pro-immunostimulant into an active immunostimulant was also found to occur using -galactosidase-enriched B16 melanoma cells. In co-culture experiments with either immune cell line, -galactosidase-enriched B16 cells effected activation of bystander immune cells.