PAP immunocytochemistry with an antiserum against serotonin (5-HT)-glutaraldehyde-protein conjugate (kindly donated by M. Geffard) was used to analyze the ultrastructural relationships of 5-HT axon terminals (varicosities) in the frontal (Fr1-Fr2), parietal (Par1), and occipital (Oc1M-Oc2) cortex of adult rats. One hundred-forty-five immunostained varicosities from Fr1-Fr2 (54 from layers I-II; 91 from layer VI) and 97 each from the upper layers (I-II) of Par1 and OcM1-Oc2 were examined in groups of serial thin sections (mean number of sections in series: 3.2 to 7.3). These terminals were of comparable shape and size in the 4 cortical sectors examined, and averaged 0.66 +/- 0.2 microns in mean diameter. The proportion of varicosities engaged in synaptic contact was evaluated by linear transformation of the relationship between the frequency of observed synaptic junctions and the number of thin sections available for examination. Reliability of the sampling was evidenced by a high coefficient of correlation (r greater than 0.95) in each cortical sector. The synaptic incidence extrapolated for whole varicosities ranged from 28% (layer VI of Fr1-Fr2) to 46% (Par1), without statistically significant differences between the 4 sectors examined. The interregional mean could thus be evaluated at 38%. The synaptic 5-HT terminals always made asymmetrical junctions, which were exclusively found on dendritic spines and shafts, and appeared more frequent on spines than shafts in the deep frontal and the upper occipital cortex. In all 4 sectors, dendritic shafts and spines and other axonal varicosities were frequently encountered in the immediate microenvironment of the immunostained varicosities. It is concluded that the cortical 5-HT innervation is predominantly nonjunctional throughout the neocortex of the adult rat, which reinforces earlier views of a highly divergent afferent system with particular functional properties and perhaps capable of widespread, global and/or sustained influences in this part of the brain.