Using whole-cell patch-clamp recordings from spinal cord slices of young (10–15 days old) rats, we have characterized and compared the properties of inhibitory synaptic transmission in lamina II and laminae III–IV of the dorsal horn, which are involved in the processing of nociceptive and non-nociceptive sensory information, respectively. All (100%) of laminae III–IV neurons, but only 55% of lamina II neurons, received both γ-aminobutyric acid (GABA)ergic and glycinergic inputs. The remaining 45% of lamina II neurons received only GABAergic synapses. Neurons receiving only glycinergic synapses were never observed. Among the 55% of lamina II neurons receiving both GABAergic and glycinergic inputs, all displayed a small proportion (∼10%) of mixed miniature inhibitory postsynaptic currents (mIPSCs), indicating the presence of a functional GABA/glycine co-transmission at a subset of synapses. Such a co-transmission was never observed in laminae III–IV neurons. The presence of mixed mIPSCs and the differences in decay kinetics of GABAA-type receptor mIPSCs between lamina II and laminae III–IV were due to the endogenous tonic production of 3α5α-reduced steroids (3α5α-RS) in lamina II. Stimulation of the local production of 3α5α-RS was possible in laminae III–IV after incubation of slices with progesterone, subcutaneous injection of progesterone or induction of a peripheral inflammation. This led to the prolongation of GABAergic mIPSCs, but failed to induce the appearance of mixed mIPSCs in laminae III–IV. Our results indicate that, compared with lamina II, inhibitory synaptic transmission in laminae III–IV is characterized by a dominant role of glycinergic inhibition and the absence of a functional GABA/glycine co-transmission.