Positive modulation of the GABA_B receptor (GABA_BR) represents a potentially useful therapeutic approach for the treatment of nicotine addiction. The positive allosteric modulators (PAMs) of GABA_BR GS39783 and BHF177 enhance GABA-stimulated [³⁵S]GTP γS-binding, and have shown efficacy in a rodent nicotine self-administration procedure reflecting aspects of nicotine dependence. Interestingly, the structural related analog, NVP998, had no effect on nicotine self-administration in rats despite demonstrating similar pharmacokinetic properties. Extensive in vitro characterization of GS39783, BHF177, and NVP998 activity on GABA_BR-regulated signaling events, including modulation of cAMP, intracellular calcium levels, and ERK activation, revealed that these structurally related molecules display distinct pathway-specific signaling activities that correlate with the dissimilarities observed in rodent models and may be predictive of in vivo efficacy. Furthermore, these GABA_BR allosteric modulators exhibit species-dependent activity. Collectively, these data will be useful in guiding the development of GABA_BR allosteric modulators that display optimal in vivo efficacy in a preclinical model of nicotine dependence, and will identify those that have the potential to lead to novel antismoking therapies.