The lateralization of motor speech function to the left hemisphere is supported by multiple lines of evidence, but relatively little is known about the anatomical basis of that specialization. In a preliminary study, we recently reported that area 45 of the left hemisphere (Broca's area) contained a subpopulation of magnopyramidal neurons which were significantly larger than any seen in the homotopic region of the right hemisphere (Hayes & Lewis, 1993a). In the present study we examined a larger sample of cases in order to determine how consistently this difference is present in the population, if it is specific to Broca's area or is a general feature of cortical regions mediating lateralized functions, and whether the subpopulation of large magnopyramidal neurons in left area 45 can be distinguished by their chemical phenotype. In Nissl-stained sections from 19 human brains, the mean (+/- SD) cross-sectional area of the largest layer III pyramidal neurons in area 45 was significantly (p < .0001) greater in the left hemisphere (522.1 +/- 128.3 microns2) than in the right (454.1 +/- 121.5 microns2). This interhemispheric difference appeared to be a unique characteristic of the largest neurons, since the mean size of all layer III pyramids in this area was not significantly different in the left (206.2 +/- 93.5 microns2) and right (213.3 +/- 103.9 microns2) hemispheres. In contrast to area 45, there was no interhemispheric difference in the mean cross-sectional area of the largest layer III pyramids in another lateralized region, primary motor cortex. In addition, in area 46, a region of prefrontal association cortex not known to be functionally lateralized, the mean somal size of the largest layer III pyramidal neurons was significantly (p < .001) smaller in the left hemisphere (402.4 +/- 84.9 microns2) than in the right (437.8 +/- 88.3 microns2). Finally, although the large layer III pyramids in area 45 were immunoreactive for nonphosphorylated neurofilament protein in both hemispheres, the mean cross-sectional area of the largest labeled neurons was significantly larger (p < .002) in the left hemisphere (525.2 +/- 149.0 microns2) than in the right (490.3 +/- 154.1 microns2). These findings demonstrate that layer III of Broca's area contains a distinctive subpopulation of neurons that may play an important role in the specific functional architecture of this region.