We have designed a laser cell deposition system that employs the phenomenon of laser guidance to place single cells at specific points in a variety of in vitro environments. Here, we describe the components of the system: the laser optics, the deposition chamber, the microinjection cell feeding system and our custom system control software application. We discuss the requirements and challenges involved in laser guidance of cells and how our present system overcomes these challenges. We demonstrate that the patterning system is accurate within one micrometer by repeatedly depositing polymer microspheres and measuring their position. We demonstrate its ability to create highly defined living patterns of cells by creating a defined pattern of neurons with neurite extensions displaying normal function. We found that the positional accuracy of our system is smaller than the variations in cell size and pattern disruptions that occur from normal cell movement during substrate adhesion. The laser cell deposition system is a potentially useful tool that can be used to achieve site- and time-specific placement of an individual cell in a cell culture for the systematic investigation of cell-cell and cell-extracellular matrix interactions.