We examined the possibility of a differential spatial control in the endogenous production of 3alpha5alpha-reduced steroids and its consequences on GABA(A) receptor-mediated miniature IPSCs (mIPSCs) in laminas II and III-IV of the rat spinal cord dorsal horn (DH). Early in postnatal development [younger than postnatal day 8 (P8)], mIPSCs displayed slow decay kinetics in laminas II and III-IV resulting from a continuous local production of 3alpha5alpha-reduced steroids. This was mediated by the tonic activity of the translocator protein of 18 kDa (TSPO), which controls neurosteroid synthesis by regulating the transport of cholesterol across the mitochondrial membrane system. TSPO activity disappeared in laminas III-IV after P8 and was functionally downregulated in lamina II after P15, resulting in a marked reduction of mIPSC duration in these laminas. TSPO-mediated synthesis of 3alpha5alpha-reduced steroids was spatially restricted, because, at P9-P15, when their production was maximal in lamina II, no sign of spillover to laminas III-IV was apparent. Interestingly, after P8, the enzymes necessary for the synthesis of 3alpha5alpha-reduced steroids remained functional in laminas III-IV and could produce such steroids from various precursors or after a single subcutaneous injection of progesterone. Moreover, induction of an acute peripheral inflammation by intraplantar injection of carrageenan, restored a maximal TSPO-mediated neurosteroidogenesis in laminas III-IV. Our results indicate that the decay kinetics of GABA(A) receptor-mediated mIPSCs in the DH of the spinal cord are primarily controlled by 3alpha5alpha-reduced steroids, which can be produced from circulating steroid precursors and/or in a spatially restricted manner by the modulation of the activity of TSPO.