During development, the sympathetic neurons innervating sweat glands undergo a neurotransmitter switch from noradrenergic to cholinergic between postnatal day (P) 4, when the sympathetic neurons first contact the sweat glands, and P21. Several in vitro experiments suggest that norepinephrine (NE), produced by sympathetic neurons, stimulates sweat glands to produce a factor that then induces the phenotypic switch. We tested this hypothesis in vivo using dopamine β-hydroxylase-deficient mice (DBH −/−), which are unable to synthesize NE and epinephrine, and tyrosine hydroxylase-deficient mice (TH −/−), which are unable to synthesize any catecholamines. The cholinergic agonist pilocarpine and electrostimulation of the sciatic nerve both elicited a sweat response in adult DBH −/− mice that was indistinguishable from the response of controls, and the cholinergic antagonist atropine effectively blocked these responses. We did note, however, a 1- to 2-week delay in the acquisition of the sweat response in DBH −/− mice. Although diminished in magnitude, a sweat response to pilocarpine was also noted in TH −/− mice at P21. Immunohistochemistry demonstrated that TH and vasoactive intestinal peptide were detectable at P14 and increased to adult levels by P21 in DBH +/− and DBH −/− mice. These observations indicate that NE is not essential for the acquisition of the cholinergic phenotype, but it may facilitate its postnatal development.