We studied μ-opioid transmission in acute slices of rat neocortex using whole-cell recordings and single-cell reverse transcription–polymerase chain reaction. The μ-opioid receptor (MOR) was found in γ-aminobutyric acidergic (GABAergic) interneurons that were either layer I cells frequently expressing neuropeptide Y or layers II–V cells expressing vasoactive intestinal peptide and enkephalin (Enk). We found that μ-opioid agonists inhibit these interneurons that are selectively excited by nicotinic agonists. The extensive overlap of μ-opioid and nicotinic responsiveness allowed μ-opioid agonists to inhibit nicotinic excitation of responsive interneurons and of their GABAergic output onto pyramidal cells. Finally, nicotinic stimulation resulted in a dynamic sequence where GABAergic transmission was first enhanced and then depressed below its baseline. This latter disinhibitory effect was prevented by a μ-opioid antagonist, indicating that excitation of nicotinic-responsive interneurons induced the release of endogenous Enk, which in turn led to MOR activation. Our results suggest that neocortical μ-opioid transmission acts as an inhibitory feedback onto nicotinic-responsive interneurons, which may change network excitability and inhibition patterns during cholinergic excitation.