Patterned neuronal cultures could be produced by plating cells dissociated from rat cortices on glass coverslips, the surface of which was printed with poly-L-lysine (PLL)-positive micropatterns. Large cell aggregates, which greatly disrupted the patterned distribution of neurons, were also generated. To investigate how large cell aggregates were formed, dissociated rat cortical neurons were plated on PLL-coated coverslips in a Petri dish, the surface of which was non-adherent to cells. The cell and cell aggregate densities found later on the coverslip surface increased significantly when larger dishes were used. Most of the neurons not attaching to substratum were able to survive for at least 24h without entering apoptosis. During this time they formed floating spherical aggregates in the medium. These aggregates subsequently were able to attach to PLL-coated coverslips and produced large aggregates resembling those found within our patterned neuronal cultures. The results suggest a causative relationship between the generation of large numbers of neurons unattached to substratum and the formation of large cell aggregates on the patterned neuronal cultures. It was further demonstrated here that patterned neuronal cultures free of large cell aggregates could be prepared by a procedure employing both stencil patterning and microcontact printing technologies.