Despite the emerging evidence to suggest that glucose-stimulated insulin secretion [GSIS] requires membrane targeting of specific small G-proteins [e.g., Rac1], very little is known with regard to the precise mechanisms underlying subcellular trafficking of these proteins in the glucose-stimulated islet cell. We previously reported activation of small G-proteins by biologically active lipids via potentiation of relevant GDP/GTP exchange activities within the cell. Herein, we studied putative regulatory roles for these lipids in the trafficking and membrane association of Rac1 in cell-free preparations derived from INS 832/13 cells. Incubation of INS 832/13 cell lysates with polyphosphoinositides [e.g., PIP₂], phosphatidic acid, phosphatidylcholine and phosphatidylserine significantly promoted trafficking of cytosolic Rac1 to the membrane fraction. Lysophosphatidic acid, but not lysophosphatidylcholine or lysophosphatidyl-serine, also promoted translocation and membrane association of Rac1. Arachidonic acid, diacylglycerol; calcium or cAMP failed to exert any clear effects on Rac1 translocation to the membrane. Together, our findings povide the first direct evidence in support of our recent hypothesis [Diabetes. 54:3523-3529, 2005], which states that generation of biologically active lipids, known to occur in the glucose-stimulated cell, may mediate targeting of Rac1 to the membrane for optimal interaction with its putative effector proteins leading to GSIS.