Nucleus raphé obscurus (NRo) modulates hypoglossal (XII) nerve motor output in the in vitro transverse brain stem slice of neonatal rats (1-5 days old); chemical ablation of NRo and its focal CO(2) acidification modulated the bursting rhythm of XII nerves. We microinjected a 4.5 mM solution of kainic acid into the NRo to disrupt cellular activity and observed that XII nerve activity was temporarily abolished (n = 10). We also microinjected CO(2)-acidified (pH = 6.00 +/- 0.01) artificial cerebrospinal fluid (aCSF) into the NRo (n = 6), the pre-Bötzinger complex (PBC) (n = 6), as well as a control region in the lateral tegmental field equidistant to NRo, PBC, and the XII motor nuclei (n = 12). CO(2) acidification of the control region had no effect on XII motor output. CO(2) acidification of the NRo significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 77%; integrated burst amplitude and burst duration increased by 64% and 52%, respectively. CO(2) acidification of the PBC significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 65%, but neither integrated burst amplitude nor burst duration changed. These results demonstrate that chemical ablation of the NRo can abolish XII nerve bursting rhythm and that stimulation of the NRo with CO(2)-acidified aCSF can excite XII nerve bursting activity. From these observations, we conclude that, in transverse brain stem slices, the NRo contains pH/CO(2)-sensitive cells that modulate XII motor output.