The developmental pattern and distribution of peptide-containing neurons in the rat heart right atrium has been studied by indirect immunofluorescence. Antibodies against neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal polypeptide (VIP) were applied to whole-mount stretch preparations of the right atria from hearts of newborn to 40 day-old animals. NPY-like immunoreactivity (L1) was compared with the synaptic vesicle marker SV2 in double immunoincubation studies. The distribution of immunofluorescence was studied by confocal laser scanning microscopy. NPY-L1 and SP-L1 were present throughout the atria already at birth, in contrast to VIP-L1 that was observed at day 10. The postnatal changes of innervation were basically quantitative, with an increase in density of nerve fibres and number of varicosities, while the basic pattern of innervation was essentially established during the first 1-10 days. NPY- and SP-positive bundles of fibres appeared to enter the right atrium along the superior caval vein, having extrinsic origins. Nerve fibres with NPY-L1 colocalized in most nerve terminals with SV2-L1, and showed a developmental pattern similar to that observed for adrenergic neurons earlier. These NPY/SV2 positive fibres probably represent the extrinsic NPY innervation. In addition, NPY-L1 was identified in large intrinsic nerve cells bodies located near the atrioventricular (AV) region. Most of the VIP-L1 was observed in short nerve fibres originating in intrinsic VIP-positive cell bodies, but a few apparently extrinsic VIP-positive fibres were found, probably representing preganglionic parasympathetic neurons. SP in the atria was probably of extrinsic (sensory) origin and no nerve cell bodies with SP-L1 were detected. The results show that the peptidergic innervation in the developing rat right atrium involves both extrinsic and intrinsic peptidergic neurons which may participate in the regulation of neurotransmission in local neuronal circuits.