In the absence of external stimuli, the mammalian neocortex shows intrinsic network oscillations. These dynamics are characterized by translaminar assemblies of neurons whose activity synchronizes rhythmically in space and time. How different cortical layers influence the formation of these spontaneous cellular assemblies is poorly understood. We found that excitatory neurons in supragranular and infragranular layers have distinct roles in the regulation of intrinsic low-frequency oscillations in mice in vivo. Optogenetic activation of infragranular neurons generated network activity that resembled spontaneous events, whereas photoinhibition of these same neurons substantially attenuated slow ongoing dynamics. In contrast, light activation and inhibition of supragranular cells had modest effects on spontaneous slow activity. This study represents, to the best of our knowledge, the first causal demonstration that excitatory circuits located in distinct cortical layers differentially control spontaneous low-frequency dynamics.