In spite of continuing controversy on the precise function of the calcium-binding proteins expressed in the hippocampal formation, nothing is known about their prenatal development in primates. In this study, calbindin-D28K, calretinin, and parvalbumin were localized in the hippocampal formation of seven rhesus monkey fetuses aged E47 to E90 (term 165 days). All of the three markers were expressed during the first half of gestation in distinct subsets of nonpyramidal neurons: calretinin-containing cells were the most numerous and relatively differentiated contrasting with a more restricted, less mature, parvalbumin-labeled population and a poor calbindin-positive nonpyramidal contingent. The granule cells and pyramidal neurons were calbindin-positive, including the pyramids of CA3 and the subicular complex, in contrast to the situation found in the adult monkey. The presubiculum and retrosplenial cortex, whose merging formed the caudal pole of the hippocampal formation, also expressed precociously the three calcium-binding proteins. A heterogeneous population of Cajal-Retzius-like cells was demonstrated in the marginal zone of the ventral hippocampal formation. The majority co-expressed calbindin-D28K and calretinin and displayed acetylcholinesterase activity but no GABA-like immunoreactivity. Major intrinsic and extrinsic pathways of the hippocampal system (mossy fiber system, alveus, fimbria, angular, and cingular bundles) were immunoreactive for calretinin and/or calbindin. The distinct developmental time course and regional pattern of distribution of calbindin-D28K, calretinin, and parvalbumin in the nonprincipal neurons suggests a precocious but asynchronous prenatal development of different inhibitory circuits in the hippocampal formation of primates. The labeling of several fiber systems in keeping with comparable early events in the entorhinal cortex (Berger et al.: Hippocampus 3:279-305, 1993), suggests the possibility of earlier functional circuits than hitherto inferred from the observations available in rodents, a hypothesis that deserves further investigation.