Anesthetic steroids with actions at GABA_A receptors may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA_A receptors. The hydrophobicity ranking predicted relative potency of GABA_A receptor potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by -cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of non-specific accumulation. An NBD-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster non-specific accumulation and departitioning than a fluorescent analogue of the high-lipophilicity (3,5)-3-hydroxypregnan-20-one (C17-NBD-35A). These differences were paralleled by differences in potentiation of GABA_A receptor function. The enantiomer of C17-NBD-35A, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-35A, but was inactive at GABA_A receptors. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore, both specific (e.g., fulfilling pharmacophore requirements) and non-specific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.