Single-particle tracking experiments were carried out with gold nanoparticle-labeled solid supported lipid bilayers (SLBs) containing increasing concentrations of ganglioside (GM(1).) The negatively charged nonoparticles electrostatically associate with a small percentage of positively charged lipids (ethyl phosphatidylcholine) in the bilayers. The samples containing no GM(1) show random diffusion in 92% of the particles examined with a diffusion constant of 4.3(+/- 4.5) x 10(-9) cm(2)/s. In contrast, samples containing 14% GM(1) showed a mixture of particles displaying both random and confined diffusion, with the majority of particles, 62%, showing confined diffusion. Control experiments support the notion that the nanoparticles are not associating with the GM(1) moieties but instead most likely confined to regions in between the GM(1) clusters. Analysis of the root-mean-squared displacement plots for all of the data reveals decreasing trends in the confined diffusion constant and diameter of the confining region versus increasing GM(1) concentration. In addition, a linearly decreasing trend is observed for the percentage of randomly diffusing particles versus GM(1) concentration, which offers a simple, direct way to measure the percolation threshold for this system, which has not previously been measured. The percolation threshold is found to be 22% GM(1) and the confining diameter at the percolation threshold only similar to 50 nm.