In this work we have analyzed the contribution of factors others than T3 itself, that might be involved in the regulation of gene expression and that may play diverse roles in vivo. This experimental set up allowed us to confirm that genes which expression in the mouse cerebral cortex is strongly dependent on thyroid hormones are indeed cellular targets of T3. The altered expression previously observed in vivo after induction of hypothyroidism was not due to a distal effect of the hypothyroid condition, but reflected a cellular effect of the hormone. The cellular system employed in this work is based on the culture of primary cells isolated from the embryonic mouse brain. These cultures are enriched in neurons but also contain astrocytes and unidentified cells. Therefore any effect observed by hormonal manipulations of these cells could have its origin in neurons or in astrocytes, depending among other factors on the relative expression of the target gene in each of these cell types. First we analyzed the specific contribution of the TRa1 and TRb receptor subtypes by measuring the response to T3 of primary cells derived from the cerebral cortex of TRa1 and TRb KO mice. The results confirm that at the cellular level both TRa1 and TRb mediate the effects of T3, with two exceptions, Dio3 and Aldh1a1. Dio3 was already shown to be regulated specifically by TRa1, and we confirm this fact in the cultured neurons. Also Aldh1a1 appears to be regulated specifically by TRa1, since no induction by T3 was observed in cells derived from TRa1 KO mice. Klf9 and Aldh1a3 show no preference for each of the TR subtypes. A recent global analysis of TR specificity in HeLa cells expressing exogenous TRa1 or TRb1 concluded that there are no complete TR subtype specificity, although TRa1 or TRb1 showed some gene preferences, depending on the time of exposure and the dose of T3. In established neural cell lines expression TRa1 or TRb1 lead to substantial differences in the gene network regulated by T3, without correlation with differential chromatin occupancy. Direct transcriptional regulation by T3, has previously been shown for Hr, Klf9 Dio3, and Shh. Here we confirm that also in Y-27632 cerebrocortical primary cells Hr, Klf9, and Shh are direct transcriptional targets of T3, given that the effect of the hormone was not blocked by previous treatment with CHX to inhibit protein synthesis. We could not determine whether Dio3 was also transcriptionally regulated by T3 because it was not stimulated by T3 at 6 hours of incubation, preventing to test the effect of CHX. However, a specific TRa1 binding site has been demonstrated in the upstream region of the gene. The fact that we could not determine whether the effect of T3 was direct, suggest the possibility that the full effect of T3 requires interaction with other intermediate proteins. Dio3 is a transcriptional target of Shh which, as mentioned above is a direct T3 target. This raises the possibility that rapid and full induction of Dio3 by T3 is the result of a direct transcriptional effect of TRa1 potentiated by the T3- dependent accumulation of Shh protein.