The α4 subunit of the GABA A receptor (GABAR) has relatively low expression in the CNS, but is increased in vivo following 48 h administration of the GABA-modulatory steroid 3α-OH-5α[β]-pregnan-20-one (THP or [allo]pregnanolone) to female rats. The purpose of the following study was to determine the optimal conditions for steroid-induced upregulation of α4 expression in an in vitro model. To this end, we used the IMR-32 cell, a neuroblastoma cell line, which normally expresses α4 mRNA at low levels. In undifferentiated IMR-32 cells, 48 h administration of THP increased α4 expression when ambient THP levels were reduced by the 5α-reductase blocker 4MA, suggesting that the background steroid milieu affects steroid regulation of this subunit. Following neuronal differentiation in serum-free medium, 48 h THP treatment significantly increased α4 expression two-fold following application of nerve growth factor (NGF) suggesting that development of neuronal processes facilitates this effect of the steroid. In the absence of NGF treatment, combined administration of 17β-estradiol (E 2) plus THP also increased α4 expression to a similar extent as THP following NGF treatment. In addition, E 2 alone effectively increased α4 expression to maximal levels following NGF treatment. In contrast, neuronal differentiation in the absence of serum deprivation did not increase α4 levels. These results suggest that both THP and E 2 can increase expression of the GABAR α4 subunit, but that this effect is dependent upon the background steroid milieu as well as the degree of neuronal development. These findings demonstrate optimal conditions for steroid-induced upregulation of the α4 subunit in an in vitro system.