A series of analogues of the neuroactive steroids 3α-hydroxy-5α- pregnan-20-one and 3α-hydroxy-5β-pregnan-20-one were studied to elucidate the mode of binding of 5α- and 5β-reduced steroids to steroid binding sites on GABA(A) receptors. Analogues which were either 3α-hydroxy-20-ketosteroids or 3α-hydroxysteroid-17β-carbonitriles and which contained various methyl group substitution patterns at C-5 and C-10 were prepared. Evaluations utilized whole-cell patch clamp electrophysiological methods carried out on cultured rat hippocampal neurons, and the results obtained with the rigid 17β-carbonitrile analogs were analyzed using molecular modeling methods. The molecular modeling results provide a rationale for the observation that the configuration of the hydroxyl group at C-3 is a greater determinant of anesthetic potency than the configuration of the A,B ring fusion at C-5. The electrophysiological results identify steric restrictions for the space that can be occupied in 5α- and 5β-reduced steriod modulators of GABA(A) recepters in the regions of space proximate to the steroid C-5, C-10, and possibly C-4 positions. This information is useful for the development of nonsteroidal analogues that can modulate GABA(A) receptors via interactions at steroid binding sites.