The stability of dendritic spines in the neocortex is profoundly influenced by sensory experience, which determines the magnitude and pattern of neural firing. By optically manipulating the temporal structure of neural activity in vivo using channelrhodopsin-2 and repeatedly imaging dendritic spines along these stimulated neurons over a period of weeks, we show that the specific pattern, rather than the total amount of activity, determines spine stability in awake mice.