Urokinase plasminogen activator (uPA) receptors (uPAR) can be engaged for activation signaling either by aggregation or by binding exogenous uPA. These signaling mechanisms require uPAR to associate with two distinct adhesion proteins, L-selectin and complement receptor 3 (CR3), respectively. uPAR contains a glycosylphosphatidylinositol anchor, suggesting that it is concentrated within glycosphingolipid-enriched microdomains, or "lipid rafts". This study was undertaken to determine the extent to which uPAR-mediated signaling is compartmentalized to lipid rafts. Human neutrophil uPAR was cross-linked or stimulated with uPA after pretreatment with the lipid raft-disrupting agents, methyl-beta-cyclodextrin or filipin III. Both agents suppressed increases in intracellular Ca(2+) concentrations ([Ca(2+)](i)) triggered by cross-linking, but did not affect [Ca(2+) ](i) in response to uPA. Neutrophil membranes were separated into lipid raft and non-raft fractions, revealing the presence of uPAR and L-selectin, but the virtual absence of CR3 alpha chain in lipid rafts, either constitutively or in response to uPAR aggregation. Fluorescence resonance energy transfer experiments confirmed close proximity of a lipid raft marker to both uPAR and L-selectin, but not CR3. We conclude that uPAR can engage distinct signaling pathways involving different partner proteins that are functionally and physically segregated from one another in both lipid raft and non-raft domains of the plasma membrane.