This paper presents a novel method for cell positioning on a substrate which combines the optical quality of glass and the cell-repelling property of fluoropolymers. The process employs plasma lithography, which utilizes the high-resolution patterning of photolithography along with the versatility of the plasma polymerization. When mammalian cells were grown over these substrates, they avoided the fluoropolymer regions and grew almost exclusively within the exposed glass areas (windows). The patterned surface reproduces the initial design of the mask, offering the possibility to control cell distances and interactions with a versatile arrangement whilst keeping the optical quality of glass for microscopy observation, in particular, when a pristine substrate in needed. This approach opens up possibilities for analysis of biological processes, such as studying cell interactions, with the integration of optical or electrical sensors. © 2009 Springer-Verlag.