The classical cytoarchitectonic maps of human prefrontal areas produced by various cartographers in the early part of this century, though similar in gross topography, differ from one another in their descriptions of the size, shape, and precise location of specific regions within the frontal promontory. The current advances in human neurobiology stimulated us to reinvestigate the cytoarchitecture of the human prefrontal cortex, beginning with areas 9 and 46, to establish a set of objective cytometric criteria for identification of these areas. Nisslstained and Gallyas-stained celloidin-embedded sections were prepared from the left hemispheres of 17 human subjects 23-73 years old, without history of neurological disease. In eight cases, light microscopic observations were supplemented by morphometric data collected on a research microscope equipped with differential interference contrast optics and interfaced to a TV monitor with video mixing equipment and a microcomputer. We used the three-dimensional counting method of Williams and Rakic (1988) to measure (1) total cortical and relative laminar thickness, (2) neuronal packing density per 0.001 mm3 in individual cortical layers, and (3) sizes of neuronal somata in selected cortical layers. Light microscopic analysis confirmed that the cortical layers are more differentiated in area 46 than in area 9, particularly at the borders of layer IV. Layers III and V exhibit clearer sublamination in area 9, while layer IV is also somewhat wider in area 46 than in area 9 (9.3% vs 6.4% of cortical thickness); the overall thickness of the cortex is the same in both areas. Cytometric analysis revealed that layer IV neurons of area 46 are more densely packed than those in area 9 (55.38 +/- 7.26 vs 45.80 +/- 4.45 neurons/0.001 mm3), as are neurons in the supragranular layers II and III combined (53.51 +/ 6.33 vs 45.69 +/ 3.81 neurons/0.001 mm3). Finally, neurons in area 46 are more homogeneous in size than those in area 9. Differences in myeloarchitecture are also evident: each area contains numerous, well-stained radial striae and two pronounced bands of horizontal fibers, but in general, area 46 is less myelinated than area 9. Objective cytometric methods can clearly distinguish two adjacent areas within the human prefrontal lobe. These findings may prove useful in the areal parcellation of the human cerebral cortex as well as provide a baseline for analysis of pathological changes in neurological and psychiatric disorders such as a schizophrenia, Huntington's or Alzheimer's diseases.