Serotonin (5-HT) nerve terminals, specifically labeled with [3H]5-HT have been counted in light microscope radioautographs from the fronto-parietal neocortex of adult rats, following prolonged superfusions with relatively high concentrations of tracer (10(-5) M or 10(-4) M), and after 15 or 30 days of radioautographic exposure. Comparative analysis of the results indicated that all 5-HT varicosities did not possess the same capacity to accumulate the exogenous amine. Nevertheless, superfusions with 10(-4) M[3H]5-HT provided a sufficient loading of 5-HT boutons to allow their complete detection within any given layer of the neocortex. The labeled varicosities were found to be present within all cortical layers, except layer VI. Their total number was extrapolated to be approximately 1 X 10(6)/cu.mm of cortex. This figure represented a mean incidence of one 5-HT nerve terminal/850-1400 cortical synapses (0.07-0.12%). The intralaminar density of 5-HT innervation increased progressively from layer V to layer I, in a distribution pattern suggestive of unspecific afferents. Based on endogenous 5-HT levels of 346 +/- 32 ng/g for the fronto-parietal neocortex, the mean 5-HT content per varicosity could be estimated to be 0.35 X 10(-3) pg, and the concentration 1900 ng/g wet weight of terminal or 0.2%. In view of their particular ultrastructural features7, ubiquitous repartition and endogenous amine content, 5-HT nerve endings may be considered as capable of exerting a rather widespread influence in the neocortex.