The horizontal cells are known to form a mono-layered mosaic in the adult retina, but are scattered at different retinal depths in early development. To help clarifying when and which spatial constraints appear in the relative positioning of these cells, we have performed a quantitative analysis of the three-dimensional (3D) organization of the horizontal cell mosaic at different developmental stages in the postnatal rat retina. We first analyzed the two-dimensional (2D) distribution of the horizontal cell projections onto a plane parallel to the upper retinal surface in retinal flat-mounts, and thus to the future mature horizontal cell mosaic. We found that this 2D distribution was non random since postnatal day 1 (P1), and had a subsequent stepwise improvement in regularity. This preceded the alignment of cells in a single monolayer, which was observed on P6. We then computed true horizontal cell spacing in 3D, finding non-random 3D positioning already on P1. Simulation studies showed that this order might simply derive from the 2D order observed in the projections of the cells in flat-mount, combined with their limited spread in retinal depth. Throughout the period analyzed, the relative positions of horizontal cells are in good agreement with a minimal spacing rule in which the exclusion zone corresponds to the average size of the inner core of the cell dendritic tree estimated from P1 samples. These data indicate the existence of different phases in the process of horizontal cell 3D spatial ordering, supporting the view that multiple mechanisms are involved in the development of the horizontal cell mosaic.