Slice preparations from neonatal rat medulla that generate respiratory rhythm in vitro were used to test for Gi/o protein-mediated mechanisms affecting breathing rhythm in mammals. The frequency of inspiratory motor discharge recorded from hypoglossal (XII) nerve roots decreased with bath application of gamma-aminobutyric acid (GABA) and norepinephrine, as well as agonists specific for GABAB, alpha 2-adrenergic, and mu-opioid receptors; 5-hydroxytryptamine had little effect on frequency. Microinjection of these specific agonists into the pre-Bötzinger complex, the site of respiratory rhythm generation in vitro, also decreased frequency. In contrast, substance P (SP) increased frequency when it was bath applied or microinjected into the pre-Bötzinger complex. To test for involvement of Gi/o proteins, pertussis toxin (PTX) was injected into the cerebrospinal fluid of newborn rats, and slices from these animals were tested 48 h later for block of drug effects on rhythm. In PTX-treated slices the frequency decrease due to GABAB, mu-opioid, and alpha 2-adrenergic receptor activation was attenuated (P < or = 0.05), whereas the SP receptor-mediated response was unaltered. To test for involvement of K+ conductances linked to Gi/o proteins Ba2+ (0.2 mM) was added to the bath before application of drugs. Ba2+ attenuated the decrease in frequency associated with GABAB (P < or = 0.05) and mu-opioid (0.10 < or = P < or = 0.05) receptor activation, whereas the alpha 2-adrenergic and SP responses were unaltered. We conclude that GABAB and mu-opioid, but not alpha 2-adrenergic and SP, receptor activation modulates respiratory frequency via a Gi/o protein-dependent Ba(2+)-sensitive ionic conductance mechanism on neurons within the medullary locus for rhythm generation. This mechanism may be a convergent pathway for control of respiratory frequency.