Along with subplate neurons, Cajal-Retzius cells (CRc) are the first neurons to be generated in the cortical anlage. Studies of their chemical content, such as neurofilament and calcium-binding proteins, might give indications on their role in cortical development at midgestation in human fetuses (20-24 gestation weeks), when the CRc are morphologically mature. Cajal-Retzius cells were immunolabeled with antibodies to subunits of neurofilament proteins SMI31 and SMI32. The SMI32 antibodies (directed against the nonphosphorylated epitope) specifically labeled the CR cell bodies, dendrites, and proximal axons in a Golgilike fashion. Specific acetylcholinesterase activity is known to be typical of all the CRc, and double labeling for SMI32 immunoreactivity and acetylcholinesterase histochemistry demonstrated that all the CRc exhibited SMI32 immunoreactivity. The SMI31 antibodies (directed against the phosphorylated epitope) exclusively labeled the CRc axons, forming a dense positive network in the deep one-half of layer 1. This plexus was much denser than the one described with the Golgi method (Marin-Padilla, 1990: J. Comp Neurol 239:89-105). Calbindin D28k, parvalbumin, and calretinin immunoreactivities were visualized in the CRc. Double-labeling experiments showed that most of the CRc contained both calbindin and calretinin and sometimes parvalbumin. These colocalizations revealed a chemical heterogeneity within the CRc population even though they were described as morphologically homogeneous. These colocalizations of calcium-binding proteins in the CRc differed from the other nonpyramidal cortical neurons where calbindin, calretinin, and parvalbumin are contained in different (mutually exclusive) neuronal populations. Based on the morphological features and differential chemical contents described for the CRc, different hypotheses on their possible role and fate are discussed.