The level of excitability of cortical neurons depends on the balance between their excitatory and inhibitory inputs (excitation/inhibition [E/I] balance). In the cortex, the E/I balance received by a neuron is dynamically maintained through a coordinated regulation of the strength of these inputs, described in term of homeostatic plasticity. Using a method allowing the determination of the E/I balance in rat cortical layer 5 pyramidal neurons (L5-PNs, the main output stage of the cortex), while keeping the interactions between excitatory and inhibitory networks functional, we examined the effects of high or low frequency of stimulation (HFS or LFS) protocols in layer 4 (in order to mimic thalamo-cortical entries) on the E-I level of the neuronal network. We previously showed that the E/I balance of L5-PNs remains stable due to a dual potentiation or dual depression of E and I after HFS or LFS protocols. Here, using a specific neuronal nitric oxide synthase (nNOS) inhibitor, we show that the related potentiation or depression of E and I (underlying homeostatic plasticity processes) required nNOS activation. We also show that application of an unspecific blocker of nitric oxide synthase (NOS) or a nitric oxide (NO) scavenger induces an increase of the E/I balance suggesting a role for a tonic NO synthesis in the regulation of the network activity. It is concluded that, in the cortex, a phasic NO effect (due to activation of nNOS) is required for the induction of homeostatic plasticity processes whereas a tonic NO signal is involved in the regulation of a set-point value for the E/I balance.