Directed control of neuronal migration, facilitating the correct spatial positioning of neurons, is crucial to the development of a functional nervous system. An understanding of neuronal migration and positioning on patterned surfaces in vitro would also be beneficial for investigators seeking to design culture platforms capable of mimicking the complex functional architectures of neuronal tissues for drug development as well as basic biomedical research applications. This study used coplanar self-assembled monolayer patterns of cytophilic, N-1[3-(trimethoxysilyly)propyl] diethylenetriamine (DETA) and cytophobic, tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (13F) to assess the migratory behavior and physiological characteristics of cultured neurons. Analysis of time-lapse microscopy data revealed a dynamic procedure underlying the controlled migration of neurons, in response to extrinsic geometric and chemical cues, to promote the formation of distinct two-neuron circuits. Immunocytochemical characterization of the neurons highlights the organization of actin filaments (phalloidin) and microtubules (β-tubulin) at each migration stage. These data have applications in the development of precise artificial neuronal networks and provide a platform for investigating neuronal migration as well as neurite identification in differentiating cultured neurons. Importantly, the cytoskeletal arrangement of these cells identifies a specific mode of neuronal migration on these in vitro surfaces characterized by a single process determining the direction of cell migration and mimicking somal translocation behavior in vivo. Such information provides valuable additional insight into the mechanisms controlling neuronal development and maturation in vitro and validates the biochemical mechanisms underlying this behavior as representative of neuronal positioning phenomena in vivo.