OBJECTIVE--To study the pattern of innervation of the conduction system of the neonatal heart in humans. DESIGN--A prospective analysis based on immunohistochemical and enzyme histochemical examination of newborn human hearts. SETTING--A general district hospital. MAIN OUTCOME MEASURES--Fresh necropsy tissue. MATERIAL--Hearts of three neonatal humans with no cardiac anomaly, freshly taken at necropsy. METHODS--Serial sectioning to obtain a three dimensional reconstruction of the cardiac conduction system, followed by identification of the pattern of innervation by immunohistochemical and enzyme histochemical techniques; with a panel of antisera against protein gene product (PGP) 9.5 as a general neural indicator; dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) as indicators for sympathetic neural tissue; and selected neuropeptides--namely, neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene related peptide (CGRP), and substance P (SP). Gomori's technique was used for locating cholinesterase activity. RESULTS--PGP immunoreactive (PGP-IR) nerves were present in large numbers in the sinus node, atrioventricular (AV) node, and penetrating atrioventricular bundle; in moderate numbers in the branching bundle; and occasionally in the bundle branches. Small numbers of DBH-IR and TH-IR nerves were seen in the sinus and AV nodes, mainly perivascularly; there were few in the penetrating and branching bundles and none in the bundle branches. A few perivascular NPY-IR nerves were seen only in the sinus node. VIP-IR, CGRP-IR, and SP-IR nerves were not seen. Pseudocholinesterase activity was found in the conduction tissue, whereas occasional acetylcholinesterase positive nerves were found only in the sinus and AV nodes. CONCLUSION--A considerable innervation of the human cardiac conduction system is present at birth, although, by comparison with the results of other studies on adult tissue, the mature pattern has not yet been established. Thus it is still in the process of maturation, especially with regard to the acquisition of various neurotransmitters, including the more recently described neuropeptides.