Motions of membrane-associated proteins within and between membranes are essential for many cellular functions. We describe the application of fluorescence recovery after photobleaching (FRAP) beam-size analysis to investigate the role of palmitoylation in the membrane targeting and membrane association dynamics of H-Ras. The method described distinguishes between FRAP by lateral diffusion and by cytoplasmic exchange, and enables to obtain an estimate of the membrane affinity in live cells. These studies show distinct roles for the two palmitoylation sites (Cys181 and Cys184) on H-Ras, with different effects on membrane affinity and microlocalization.