The aggregation of Gramicidin A (gA) in dipalmitoylphosphatidylcoline (DPPC) monolayers is investigated by both thermodynamic and structural methods. Compression isotherm analysis and atomic force microscopy (AFM) observations are performed. Our experimental results indicate that gA aggregation does occur in DPPC monolayers even at very low gA concentration (about 8 x 10(-4) mol%). At the low gA concentration limit, the aggregation process seems to be mainly horizontal (i.e., side-by-side, into the monolayer plane), following a fractal pattern growth producing the formation of typical, flat (0.5 nm height) "doughnut" structures, with a diameter of approximately 150 nm. These structures appear to be composed of smaller subunits (about 70 nm diameter) showing the same doughnut structure. At a molar fraction of approximately 3.8 mol%, the big doughnuts start to disaggregate and only small doughnuts appear. Above a gA concentration of approximately 4.4 mol%, all doughnuts (large and small) disappear, and the morphology assumes the appearance of a patchwork of two distinct phases: one that, being very flat, can be associated with a gA-free or gA-poor DPPC phase, and a second one, characterized by a more corrugated surface, associated with a gA-rich DPPC phase. At gA concentration of approximately 5 mol%, a percolation transition in the gA-rich DPPC phase occurs. Thermodynamic data indicate that the maximum of miscibility between gA and DPPC molecules occurs at approximately 28 mol%, suggesting that gA could aggregate in hexamers that are, on average, bound to 16 DPPC molecules. At the same concentration, AFM images show a network of small gA aggregation units of a size compatible with gA hexamers.