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The biggest problem when analyzing the brain is that its synaptic connections are extremely complex. Generally, the billions of neurons making up the brain exchange information through two types of highly specialized structures: chemical synapses (the vast majority) and so-called gap junctions (a substrate of one class of electrical synapse). Here we are interested in exploring the three-dimensional spatial distribution of chemical synapses in the cerebral cortex. Recent research has showed that the three-dimensional spatial distribution of synapses in layer III of the neocortex can be modeled by a random sequential adsorption (RSA) point process, i.e., synapses are distributed in space almost randomly, with the only constraint that they cannot overlap. In this study we hypothesize that RSA processes can also explain the distribution of synapses in all cortical layers. We also investigate whether there are differences in both the synaptic density and spatial distribution of synapses between layers. Using combined focused ion beam milling and scanning electron microscopy (FIB/SEM), we obtained three-dimensional samples from the six layers of the rat somatosensory cortex and identified and reconstructed the synaptic junctions. A total volume of tissue of approximately 4500μm3 and around 4000 synapses from three different animals were analyzed. Different samples, layers and/or animals were aggregated and compared using RSA replicated spatial point processes. The results showed no significant differences in the synaptic distribution across the different rats used in the study. We found that RSA processes described the spatial distribution of synapses in all samples of each layer. We also found that the synaptic distribution in layers II to VI conforms to a common underlying RSA process with different densities per layer. Interestingly, the results showed that synapses in layer I had a slightly different spatial distribution from the other layers. ; This work was supported by grants from the following entities: the Spanish Ministry of Economy and Competitiveness (grants TIN2013-41592-P to Laura Anton-Sanchez, Concha Bielza, and Pedro Larrañaga, BFU2012-34963 to Javier DeFelipe, and SAF 2010-18218), CIBERNED CB06/05/0066 to Javier DeFelipe, the Cajal Blue Brain Project (Spanish partner of the Blue Brain Project initiative from EPFL) to Laura Anton-Sanchez, Concha Bielza, Angel Merchán-Pérez, José-Rodrigo Rodríguez, Javier DeFelipe, and Pedro Larrañaga, and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 604102 (Human Brain Project) to Concha Bielza, Angel Merchán-Pérez, Javier DeFelipe, and Pedro Larrañaga. ; Peer reviewed ; Peer Reviewed