Descriptive studies have delineated a developmental change in neurotransmitter phenotype from noradrenergic to cholinergic in the sympathetic innervation of sweat glands in rodent footpads. Transplantation and culture experiments provide evidence that interactions with the target tissue induce this change. Recent studies with an antiserum that recognizes the vesicular acetylcholine transporter (VAChT) suggest, however, that the development of cholinergic function in sympathetic neurons, including those that innervate sweat glands, occurs prior to and does not require target contact. To clarify these apparently contradictory findings, we directly compared the appearance of VAChT immunoreactivity in the sympathetic neurons that innervate sweat glands with the time that axons contact this target. We find that VAChT immunoreactivity is not detectable in either the axons or cell bodies of sweat gland neurons until several days after target innervation. Before and during VAChT acquisition, the developing sweat gland innervation contains vesicular stores of catecholamines. An analysis of mutant mice that lack sweat glands was undertaken to determine whether VAChT expression requires target interactions and revealed that VAChT does not appear in the absence of glands. These findings, together with previous studies, confirm the target dependence of cholinergic function in the sympathetic neurons that innervate sweat glands.