Human functional neuroimaging studies have consistently shown that the superior element of the precentral sulcus (sPCS), near the caudal end of the superior frontal sulcus (cSFS), is activated during oculomotor tasks, and refer to this area as the frontal eye field (FEF). However, the anatomy of this area in humans has not been examined systematically, nor has the correspondence between traditional cytoarchitectonic maps and recent studies of the functional neuroanatomy of the FEF been determined. To identify the chemoarchitectonic features of this fMRI-defined area of the human sPCS, we labeled tissue sections of postmortem human brains containing the sPCS and the cSFS with antibodies against the neuronal nuclear protein (NeuN), the nonphosphorylated neurofilament triplet protein (NNFP), and the calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV). Distinctive chemoarchitectural features of this area of the sPCS compared with rostral and caudal regions were consistently found across subjects and consisted in clusters of large, intensely immunoreactive (IR) pyramidal cells in deep layer V and the presence of layer IV in both NNFP and NeuN-labeled material. In sections labeled for calcium binding proteins, the two walls of the sPCS were characterized by: (1) a higher density of CR-IR neurons; (2) a relative lack of CB-IR pyramidal cells; and (3) higher density of layers II-III CB-IR neurons and more large PV-IR interneurons in deep layer III. These specific chemoarchitectural features may differentiate the location of the human FEF from rostral cortical regions and support the functions for which it is specialized.