In order to explore the molecular nature and the regulation of dense cytomatrix which interconnects MT, NF and membranous organelles in neurons (9), the interactions between NF, MT and each of these cytoskelatal elements with brain mitochondria were investigated in vitro using biochemical and viophysical methods. From these studies, the following conclusions were drawn: 1‐ Pure NF form in vitro a highly viscous gel, dependent upon the phosphorylation state of the side arms of the NF‐H and M subunits which might participate directly to the interactions since antibodies specific of these phosphorylated sites inhibited efficiently the NF gelation. This process is modulated by both ATP hydrolysis and soluble molecules from nervous tissue and it might reflect the highly controled organization of NF bundles in axons. 2‐ In contrast with NF, low viscosity levels were detected in MT suspensions. However, the occurrence of weak interactions between MT were deduced from studies with taxol, ATP, AMP‐PNP and Mg ions, which affected the viscosity and the organization of MT in vitro, possibly through MAPs mediated interactions. 3‐ Mitochondria associated permanently in vitro to few MT through cross‐bridges involving MAPs, which bind to specific sites on the outer membrane (17). In addition, brain mitochondria (and not liver mitochondria) interact with NF in an ATP‐dependent manner, through thin cross‐bridges possibly involving the NF‐H and M subunits since these molecules, when purified, compete efficiently with MAPs for the binding to membrane sites. These results suggest the participation of structure MAPs and of NF‐H and M subunits in the spatial organization MT and NF and in anchoring mitochondria to the cytomatrix.