Although the entorhinal cortex is a key structure connecting the hippocampal formation with the rest of the cerebral cortex, little is known about its early chemoanatomical development in primates. In the present study, a cytoarchitectonic analysis and immunocytochemical detection of somatostatin, neurotensin, parvalbumin, calbindin-D 28K, DARPP-32, as well as tyrosine hydroxylase, dopamine-β-hydroxylase, and serotonin, were carried out on serial sections of the entorhinal cortex of six rhesus monkey fetuses aged E47 to E90 (gestation period 165 days). At E56 the cortical plate of the entorhinal cortex already exhibited a sublamination; at E64 the lamina dissecans was partly formed, allowing the emergence of the lamina principalis externa and interna, and at E83 most of the regional and laminar subdivisions characteristic of the adult cortex could be identified, except for the rhinal sulcus restricted to a small dimple. The neurochemical development paralleled the early cytoarchitectonic differentiation, both largely preceding that of the neighboring cortical areas. The somatostatin-like immunoreactive innervation, first detected at E56, was very dense as early as E64 and displayed by E83 a laminar distribution similar to that found in the adult. Labeled neurons indicated an intrinsic origin for this innervation but an extrinsic connection might be present as labeled fibers in the subplate of the entorhinal cortex were in continuity with positive fibers in the intermediate zone of the hippocampal formation. A faint neurotensin-like immunoreactivity first detected at E64 became prominent at E83 in the entorhinal cortex but stopped abruptly at the anlage of the rhinal sulcus. The lack of neurotensin-labeled neurons contrasted with their presence in other parts of the hippocampal region and suggested a precocious extrinsic connection. Only rare parvalbumin-LIR neurons were detected at midgestation, whereas calbindin-D 28K was expressed from E47 on in Cajal-Retzius cells and from E56 on in various types of neurons in the cortical plate and subplate. Most characteristic was a category of medium-sized, deeply stained calbindin-LIR neurons, present only in the lamina principalis externa and possibly corresponding to the population of large neurons described by Kostovic et al. (1990, Soc Neurosci Abstr 16:846) in early developing entorhinal cortex of human fetuses. These and probably other neurons were also DARPP-32-positive, suggesting the possibility of an early dopaminergic regulation. Indeed, the monoaminergic innervation of the entorhinal cortex was detected from E56 on and gradually increased in density, displaying areal and laminar differences in the distribution of the dopaminergic, noradrenergic, and serotoninergic afferents. Each of the neuroactive substances under study also displayed a distinct pattern with respect to the mediolateral and rostrocaudal organization of the entorhinal cortex. These findings reveal a remarkably early maturation of the entorhinal cortex during the first half of gestation, compatible with the possibility of trophic effects. However, in keeping with the early neurochemical development of the hippocampal formation, it does not seem unreasonable to suggest that some functional circuits are already forming in the hippocampo-entorhinal complex of primates during the first half of gestation.