GABA(B) receptor agonists produce hypothermia and motor incoordination. Two GABA(B(1)) receptor subunit isoforms exist, but because of lack of specific molecular or pharmacological tools, the relevance of these isoforms in controlling basal body temperature, locomotor activity, or in vivo responses to GABA(B) receptor agonists has been unknown. Here, we used mice deficient in the GABA(B(1a)) and GABA(B(1b)) subunit isoforms to examine the influence of these isoforms on both baseline motor behavior and body temperature and on the motor-incoordinating and hypothermic responses to the GABA(B) receptor agonists l-baclofen and gamma-hydroxybutyrate (GHB). GABA(B(1b))(-/-) mice were hyperactive in a novel environment and showed slower habituation than either GABA(B(1a))(-/-) or wild-type mice. GABA(B(1b))(-/-) mice were hyperactive throughout the circadian dark phase. Hypothermia in response to l-baclofen (6 and 12 mg/kg) or GHB (1 g/kg), baclofen-induced ataxia as determined on the fixed-speed Rotarod, and GHB-induced hypolocomotion were significantly, but for the most part similarly, attenuated in both GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice. We conclude that l-baclofen and GHB are nonselective for either GABA(B(1)) receptor isoform in terms of in vivo responses. However, GABA(B(1)) receptor isoforms have distinct and different roles in mediating locomotor behavioral responses to a novel environment. Therefore, GABA(B(1a)) and GABA(B(1b)) isoforms are functionally relevant molecular variants of the GABA(B(1)) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors.