Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth due to failure of respiratory movement. To clarify this reason, we examined respiratory rhythm generation of mutant fetus rats. When the fetuses were delivered at embryonic days (E) 21.5 (or E22.5, that is just before birth) by Caesarean section, rSey2/rSey2 rats did not show any ventilation by respiratory movement. However, electrophysiological analyses using brainstem-spinal cord preparations revealed that there were no significant changes in respiratory rates monitored by the fourth cervical ventral root (C4) among rSey2/rSey2, Pax6 heterozygous mutant (rSey2/+), and wild type (+/+) littermates. In addition, response of respiratory rhythm to CO2 was also similar in all genotypes. Neurons of the parafacial respiratory group (pFRG), one of the respiratory rhythm generators in the rostral medulla, express a transcription factor Phox2b. In the immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the pFRG of rSey2/rSey2 rats. Notably, the brainstem of rSey2/rSey2 at E21.5-E22.5 tended to be smaller in the size than those from rSey2/+ and +/+ littermates. Functionally, development of respiratory rhythm generation at E20.5 tended to be delayed (i.e. slow and unstable rhythm) in rSey2/rSey2 rats in electrophysiological analyses. Our findings demonstrated that rSey2/rSey2 rats possess almost normal structures and functions in the respiratory rhythm generator and central chemoreceptors of the medulla just before birth, despite tendency of slower development during the fetal period. We concluded that the failure of respiratory movement that results in lethality of rSey2/rSey2 might be due to defects in anatomical formation of cephalic, facial, or hypoglossal regions, not in the respiratory nervous system.