Preparation of nervous tissue as an organotypic culture permits examination of nervous system development and interactions between neurons and with their environment. In order to evaluate the statistical significance of morphological changes, it is necessary to quantify tissue populations and parameters. Recent advances in stereology facilitate the gathering of morphometric data, while eliminating sources of error associated with traditional methods. Unfortunately, the new stereology has been perceived as complex and, therefore, has been slow to gain acceptance within neuroscience investigations in general and in vitro applications in particular. Here, we present a detailed application of the new, unbiased stereology to organotypic hippocampal cultures. These methods allow assessment of neuronal survival and morphological plasticity to culture conditions with increasing culture age. Neuronal number was assessed from point-sampled intercepts of cells sampled without bias by the selector procedure in vertical sections through hippocampal cultures. Surface area was estimated from profile intersects of neuronal processes with a cycloid array. The reference volume was determined by the Cavalieri method; this permitted expression of results as absolute values rather than as component densities. The importance of absolute values to valid interpretation of quantitative data is demonstrated by the results for neuronal number. The methods applied here provide a powerful approach to quantitation of organotypic cultures, and represent an important extension of qualitative studies of cultured nervous tissue.