Previously, we found that the brainstem neuronal network in normal rats undergoes abrupt neurochemical, metabolic, and physiological changes around postnatal days (P) 12–13, a critical period when the animal's response to hypoxia is also the weakest. This has special implications for sudden infant death syndrome (SIDS), insofar as seemingly normal infants succumb to SIDS when exposed to respiratory stressors (e.g., hypoxia) during a narrow postnatal window. Because an abnormal serotonergic system has recently been implicated in SIDS, we conducted a large-scale investigation of the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) and serotonin transporter (SERT) with semiquantitative immunohistochemistry in multiple brainstem nuclei of normal rats aged P2–21. We found that 1) TPH and SERT immunoreactivity in neurons of raphé magnus, obscurus, and pallidus and SERT in the neuropil of the pre-Bötzinger complex, nucleus ambiguus, and retrotrapezoid nucleus were high at P2–11 but decreased markedly at P12 and plateaued thereafter until P21; 2) SERT labeling in neurons of the lateral paragigantocellular nucleus (LPGi) and parapyramidal region (pPy) was high at P2–9 but fell significantly at P10, followed by a gradual decline until P21; 3) TPH labeling in neurons of the ventrolateral medullary surface was stable except for a significant fall at P12; and 4) TPH and SERT immunoreactivity in a number of other nuclei was relatively stable from P2 to P21. Thus, multiple brainstem nuclei exhibited a significant decline in TPH and SERT immunoreactivity during the critical period, suggesting that such normal development can contribute to a narrow window of vulnerability in postnatal animals. J. Comp. Neurol. 518:1082–1097, 2010. © 2009 Wiley-Liss, Inc.