Abstract : Neocortical layer 5 pyramidal neuron (L5PN) activity must be finely tuned in cortical columns to ensure the dynamic integration of sensory inputs and brain responsiveness. This is achieved through the coordinated and balanced activity of wired L5PN-targeting excitatory (E) and inhibitory (I) neurons, leading to a 20%-80% E-I balance set-point. Any deregulation of the E-I balance or of the serotoninergic system both lead to neuronal diseases including depression and schizophrenia, but how serotonin tunes the E-I balance is still unknown. In rat visual cortex, we show that endogenous 5-HT has differential modulatory effects on the E-I balance, both between 5-HT receptors subtypes (1A, 2A, 3, 4, 7) and in relation with their specific localization in the cortical column. We reveal for the first time that serotonin finely tunes the E-I balance while keeping L5PN responsiveness within a functional range. Our results shed new light on the functional segregation of 5-HTR subtypes in sensory circuits.