Advances in neural tissue engineering have resulted in the development and implementation of three-dimensional (3-D) neural cellular constructs, which may serve as neurofidelic in vitro investigational platforms. In addition, interfacing these 3-D cellular constructs with micro-fluidic and/or micro-electrical systems has created biohybridized platforms, providing unprecedented 3-D microenvironmental control and allowing noninvasive probing and manipulation of cultured neural cells. Cells in the brain interact within a complex, multicellular environment with tightly coupled 3-D cell-cell/cell-extracellular matrix (ECM) interactions; yet most in vitro models utilize planar systems lacking in vivo-like ECM. As such, neural cultures with cells distributed throughout a thick (> 500 microm), bioactive extracellular matrix may provide a more physiologically relevant setting to study neurobiological phenomena than traditional planar cultures. This review presents an overview of 2-D versus 3-D culture models and the state of the art in 3-D neural cell-culture systems. We then detail our efforts to engineer a range of 3-D neural cellular constructs by systematically varying parameters such as cell composition, cell density, matrix constituents, and mass transport. The ramifications on neural cell survival, function, and network formation based on these parameters are specifically addressed. These 3-D neural cellular constructs may serve as powerful investigational platforms for the study of basic neurobiology, network neurophysiology, injury/disease mechanisms, pharmacological screening, or test-beds for cell replacement therapies. Furthermore, while survival and growth of neural cells within 3-D constructs poses many challenges, optimizing in vitro constructs prior to in vivo implementation offers a sound bioengineering design approach.