In order to analyze whether the bovine pineal gland is a homogeneous or a heterogeneous structure as far as monoamine content, the regional differences in norepinephrine (NE), dopamine (DA), serotonin (5HT), and 5‐hydroxyindoleacetic acid (5HIAA) contents were assessed by high‐pressure liquid chromatography. NE content was maximal in the proximal (close to the recessus pinealis) region and decreased in a rostral‐caudal direction to achieve minimal values at the distal region. DA exhibited an opposite trend to NE, NE/DA ratios varying from 3.2 (proximal region) to 1.4 (distal region). Significantly lower NE content was found at the inferior as compared to the superior pineal region, and at the cortex as compared to the medulla. No significant differences were detected in DA concentration of these latter pineal regions, or in 5HT or 5HIAA concentration as a function of the region examined. 3H‐5HT and 3H‐NE uptake were maximal at the proximal zone in a rostral‐caudal direction, at the superior as compared to the inferior region, and at the medulla as compared to the cortex. Unlabeled NE was equally effective to compete with 3H‐5HT uptake in the several pineal regions studied. While NE increased maximally 3H‐5HT release in a rostral‐caudal direction, DA exhibited an opposite trend, displaying maximal 5HT release activity at the distal pineal region. DA and NE 5HT‐releasing activity were greater in the pineal medulla than in the cortex, and did not exhibit differences in the superior as compared to the inferior pineal aspects. Excess (55 mM) K+ released 3H‐5HT to a similar extent regardless of the pineal region examined. Copyright © 1990, Wiley Blackwell. All rights reserved