The human primary auditory area (AI) corresponds to granular cortex located on Heschl’s gyrus. We studied its pattern of cytochrome oxidase and acetylcholinesterase activity in 10 normal human hemispheres. In cytochrome-oxidase-stained coronal sections layer IV was prominent by its dark staining. The overall staining intensity varied along the medio-lateral extent of AI; a 2.0–2.5-mm-wide antero-posterior dark band was present at mid-AI. In acetylcholinesterase-stained coronal sections a dark antero-posterior band appeared at the same location, corresponding to the highly granular part of AI. In cytochrome-oxidase-stained tangential sections of flattened AI, ≈ 500-μm thick alternating dark and light cytochrome oxidase stripes were present in layers III and IV. These stripes were perpendicular to the dark band. Comparison with tonotopic maps of human AI obtained by activation studies suggests that the cytochrome oxidase and acetylcholinesterase dark band is most likely parallel to isofrequency lines and may correspond to the representation of frequencies critical for speech comprehension. The narrow stripes may be related to particular binaural or ampliotopic domains, whose presence is suggested by evidence from electrophysiological recordings in cat AI and from magnetoencephalographic studies in humans.