Secreting tubules, nerves fibers, and blood vessels in human sweat glands (SGs) were fluorescently stained by immunohistochemical and lectin methods for examination with a laser scanning confocal microscope (LSCM). Using these techniques, the three-dimensional distribution of up to three substances within a single specimen was investigated by collecting a series of optical sections for each of three fluorophores. Each SG received several nerve fibers. These branched into delicate bands of one or more axons that ran longitudinal to the sweat tubule then encircled the tubule. A heavy complement of capillaries was interwoven among the sweat tubules. Sweat ducts were accompanied from the SG toward the skin surface by one or two longitudinally oriented nerve fibers and capillaries. Immunoreactive staining of nerves was heaviest with protein gene product 9.5 antibody, but triple labeling showed that immunoreactivity to calcitonin gene- related peptide, vasoactive intestinal polypeptide, and synaptophysin was also present in the same axons. Substance P-immunoreactive axons were sparse in SGs but were present in other areas of the skin. The techniques used have considerable potential in examination of human skin biopsies for diagnosis of disorders affecting the somatic and autonomic nervous systems.